O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an oral gavage study in rats (OECD 421; Reproductive/developmental screening test), the test item did not cause any toxicologically significant reproductive/developmental effect at or below 250 mg/kg bw/d, thus providing the latter as the NOAEL value (Envigo study, 2018).

Supporting studies on 2-ethylhexanol, one of the hydrolytic products of the parent substance, did not show any adverse effect on testicular damage (in vivo) and germ cell detachment in testicular cultures (in vitro), respectively. (Sjoberg et al 1986).

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

An apperently well conducted GLP study per information availablae in public domain on 2-ethylhexyl terephthalate, which was used as read across for 2-ethylhexanol, which is one of the two hydrolytic byproducts of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8), the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

As described in the study report

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain details: Crl:CD(SD)IGS BR rats
- Source: Charles River Laboratories, Raleigh, NC, USA
- Age at arrival: 42 days
- Age at study initiation: 52 days
- Weight at study initiation: data not presented in publication
- Housing:
pre -mating - F0 and F1 parental anim als were housed individually in clean, wire-mesh cages
mating: during cohabitation the pairs were left in the home cage of the male animal
after mating: after 14 days or after evidence of mating males were further caged individually in their cages until
scheduled necropsy, females were transferred to plastic maternity cages with nesting material (Bed-O Cobs). Damns
were housed there until weaning on lactation day (LD) 21
- Diet: fe d PMI Nutrition International, Inc., Certified Rodent Lab diet 5002, ad libitum
- Water: municipal water (reverse osmosis treated on-site), ad libitum
- Acclimation period: 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
As the study is performed according to guidelines appropriate conditions are assumed.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of d iet (frequency): every 2 weeks
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or until evidence of mating
- Proof of pregnancy: copulatory plug or sperm in vaginal smear referred to as day 0 of pregnancy
- When e vidence of mating did not occur within 14 days the female was plaved in a maternity cage with no further
opportunity for mating.
- After successful mating each pregnant female was caged (how): in plastic maternity cages with nesting material
(Bed-O Cobs) until LD21/PND21

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

stability, homogeneity and verification of DEHT concentration in feed was confirmed by HPLC/UVD

Duration of treatment / exposure:

Parental animals F0: total of 17 to 19 weeks
males: 70 consecutive days before mating, throughout mating until scheduled necropsy (6 to 10 days after weaning of
litte rs);
females: 70 consecutive days befo re mating, throughout mating, gestation and lactation until scheduled necropsy
Parental animals F1: according to the treatment of F0 test animals

Frequency of treatment:

daily (fed ad libitum)

Details on study schedule:

Parental animals started to receive diet containing the test material when approximately 8 weeks for the F0 generation
and at PND22 for the F1 generation. In both cases the animals were treated for 70 days pre -mating thus the F0
animals were approximately 18 weeks old when mating was started. The F1 animals were 13 weeks old when mating
was started.

Remarks:

Doses / Concentrations:
0, 3000, 6000, 10000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Tim e schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:weekly
BODY WEIGHT: Yes
- Time schedule for examinations:
F0/F1 m ales: weekly throughout the study and just befo re necropsy
F0/F1 females: weekly until evidence of copulation, then on GD (gestation day) 0, 4, 7, 11, 14, 17, 20 and on LD 1, 4,
7, 14, and 21
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/k g body
weight/day: Yes
F0/F1 males: weekly throughout the study, except during mating
F0/F1 females: weekly except during mating and after evidence of copulation, then on GD (gestation day) 0, 4, 7, 11,
14, 17, 20 and on LD 1, 4, 7, 14, and 21

Oestrous cyclicity (parental animals):

Sta rting 21 days be fore mating until the day of verified mating vaginal smears were prepared daily to determine
estrous cyclicity. Vaginal smears were carried out on females also on the day of their euthanasia to determine the
stage of estrus.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight (right and left), epididymis weight (right and left), cauda epididymis weight (right and left) sperm count in
testes, sperm count in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (equal number/sex/litter as nearly as possible); excess pups were killed

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
All pups were identified and sexed at PND0. Survival and signs of toxicity were examined twice daily, Body weight was
assessed on PND 1, 4, 7, 14, and 21. The following parameters were noted: number and sex of pups, s tillbirths, live
births, postnatal m ortality, presence of g ross anomalies, detailed physical examination
In F1 pups selected as next generation balnopreputial separation and vaginal perforation were used to assess sexual
maturation.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities (using diffe rent methods when pups died either inbetween PND0 and 4 or
after PND4)

Postmortem examinations (parental animals):

SACRIFICE
F0/F1 parental animals were sacrificed 6 to 10 days after weaning of litters. Female animals that experienced total
litter loss were euthanized within 24 hours.
F1 weanlings not selected for the next generation and F2 pups were euthanized on PND 21.
GROSS NECROPSY
- Gross necropsy was performed on all parental animals and weanlings, selected organs were weighed.
HISTOPATHOLOGY / ORGAN WEIGHTS
Selective histopathologic examination was carried out on 10 parental animals per gender in the control and high dose
groups. In addition, uteri and vaginas from all F0, and F1, adult female animals in the control and high dose groups
were examined his topathologically.
Moreover, uteri and vaginas from all F0 and F1 adult female animals in the low- and mid-dose groups with uterine
weights > 1 g were also examined.
The following organs were weighed after termination:
brain, liver, kidneys, splee,, seminal vesicles/coagulating gland, prostate, testis (right and le ft), epididymis (right and
left), cauda epididymis (right and left), uterus, ovaries, thymus gland, adrenal glands, pituitary gland
No detailed overview of tissues prepared for microscopic examination is given in the publication.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed on PND4.
- F2 offspring were sacrificed on PND21.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).
GROSS NECROPSY
- Gross necropsy was performed, but no details are given within the publication.
HISTOPATHOLOGY / ORGAN WEIGTHS
No details are given in the publication.

Statistics:

The study was performed using two -tailed analysis with a minimum significance level of 5%.
The following statis tical analyses were carried out:
- fo r parental mating and fertility indices: chi square test with Yates' correction factor
- fo r body weights (adult and pup from PND 4 to 21), feed consumption, organ weights, sperm numbers, precoital
intervals , live litter size, aquistion of developmental landmarks: one-way analysis of variance (ANOVA) with Dunnett's
test
- fo r sperm motility, sperm morphology, pup gender ratio, postnatal survival: Kruskal-Wallis test with Mann-Whitney U
test
- fo r histopathological data: Fisher's exact test
- fo r offspring weights before standardisation o n PND4: analysis of covariance and Student's t-test

Reproductive indices:

To assess reproductive parameters the following indices were given:
- estrous cycle length (days)
- precoital interval (days)
- mating index (%)
- fertility index (%)
- ge station length (days)
No details on calculation of the indices were given in the publication.

Offspring viability indices:

The following offspring indices were given:
- total pups/litter (n)
- live pups/litter (n; mean live litter size)
- ge nder ratios (% per litter)
- postnatal pup survival (% per litter) on PND 0, PND 0 to1, PND 1 to 4, PND 0 to4, PND 4 to 21
No details on calculation of the indices were given in the publication.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality:
F0:
- 10000 ppm: 3 female rats 2 to 8 days after weaning of F1 pups were found dead or were sacrificed in extremis
F1:
- 10000 ppm: 7 female rats 2 to 8 days after weaning of F2 pups were found dead or were sacrificed in extremis
Single male deaths in F0 control and mid dose group as well as in the F1 high dose group were not attributed to
substance treatment by the authors. All other F0/F1 animals survived to scheduled necropsy.
Body Weights:
F0:
- 10000 ppm:
males: reduced mean weekly bw gain (15-25%) during weeks 3 to 4, 4 to 5, and 6 to 7 resulted in a slightly reduced
bw at termination (5%) in this group
females: reduced bw gain during gestation (12%), resulting in reduced bw during gestation and throughout lactation
- 3000 and 6000 ppm: No test article-related effects on m ean weekly body weights or other parameters in correlation
to bw of male and female animals were seen.
F1:
- 10000 ppm:
males: reduced mean body weight, along with lower mean birth weights and decreased growth befo re weaning from the
F0 maternal animals, resulted in reduced mean body weights until scheduled necropsy (-13% at termination)
females: resulting from reduced mean bw during the pre-weaning period, pre-mating bw were decreased relative to the
control (statis tically significant); comparable to F0 reduced bw gain during gestation, resulting in reduced bw during
gestation and throughout lactation, final body weight - 6%
- 6000 ppm:
males reduced mean body weight, along with lower mean birth weights and decreased growth befo re weaning from the
F0 maternal animals, resulted in reduced mean body weights until scheduled necropsy (-6% at te rmination)
females: resulting from reduced mean bw during the pre-weaning period, pre-mating bw were decreased relative to the
control (not statistically significant); reduced mean bw during GD11 to 20 and LD1 to 14, final body weight - 6,5%
- 3000 ppm: No test article -related effects on m ean weekly body weights or other parameters in correlation to bw of
male and female animals were seen.
Food Consumption:
The authors calculated the mean compound consumption (fo r details see table in 'Any other information').
F0:
- 1000 ppm:
females: reduced throughout gestation and lactation (statistically significant; correlating with reduced bw)
F1:
- 10000 ppm
males: slightly reduced throughout generation (correlating with reduced bw)
females: reduced throughout gestation and lactation (statistically significant; correlating with reduced bw)
- 6000 ppm:
males: slightly reduced during the first week after weaning (correlating with reduced bw)
females: slightly reduced from PND7 to 14 (correlating with reduced bw)
There were no other treatment related changes in food consumption (g/kg/day) in the remaining male and female
tre atment groups.
Organ Weights:
F0:
- 6000 &10000 ppm: females: relative liver wt increased
F1:
- 6000 &10000 ppm: females: relative liver wt increased
Several statistically significant decreases were observed in mean absolute organ weights in the F0 and F1 adults (e .g.
spleen, kidneys, uterus, testes). In most cases, these changes disappeared when compared relative to the body
weight. This suggests the differences were due to the decreased body weights. Moreover there were no correlative
findings in the respective organs in macroscopic and microscopic examinations. Uterine weights in controls were
unusually high due to the fact that a lot of the animals were in estrous or proestrous on the day of necropsy.
Similarities between the uterine weights from F1 treatment groups with the F0 control further suggests that the effe ct is
not substance related.
Histopathology: There were no test substance -related microscopic lesions in any of the tissues examined fo r the adult
F0 and F1 rats.

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level / Remarks no reproductive toxicity was observed under the conditions of this study; corresponding to 149-205/198-450 mg 2- EH/kg bw/day in F0 males/females or 184-298/232-516 mg 2-EH/kg bw/day in F1 males/females

Dose descriptor:

NOAEL

Remarks:

for parental toxicity

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Basis for effect level / Remarks high and mid dose group: reduced m/f pup weights on PND14 to21 in F1 and F2 generation

Remarks on result:

other: Generation: F1/F2 (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Body Weights: (litter effect was accounted for: i.e. number of pups born per litter affetcs the mean pup weight: in
litters with larger number of animals the mean pup bw is lower and vice verca; litter sizes can be found in detail in 'Any
other information')
F1 (pups)
- 6000 & 10000 ppm: reduced postnatal pup body weights in e arly postnatal phases and later on a t PND14 to 21 and
pup body weight gains (decreases in feed consumption in the female animals from this group during gestation and
lactation may have contributed to the early effects, reductions in pup body weight gain later in lactation may have
been due to direct consumption of the treated feed or taste aversion to the same)
F2:
- 10000 ppm: reduced postnatal pup body weights in early postnatal phases and later on at PND14 to 21
- 6000 ppm:reduced postnatal pup body weights only at PND14 to 21
No other relevant differences in mean body weight or body weight gains were observed among any group.
Food Consumption: Not Applicable
Developmental Landmarks:
F1 (weanlings):
- 10000 ppm: approx. 2 days delay in age of acquisition of balanopreputial separation, most probably due to the
reduced body weight
- in female animals mean age of vaginal patency was similar between control group and all treatment groups.
The body weights on the day of attainment were decreased (significant statistically) at all dose levels reflecting either
the slightly younger age of the animals (3000 ppm group) or the reduced body weights (6000 and 10000 ppm
groups).
Organ Weights: all changes were noted on PND21
F1 (weanlings):
- 10000 ppm:
males: decreased relative spleen weight (13%), increased mean relative brain weight (25%)
females: increased mean relative brain weight (25%)
- 6000 ppm:
females: increased mean relative brain weight (12%)

F2:
- 10000 ppm:
males: decreased relative spleen weight (8%), increased mean relative brain weight (23-25%)
females: decreased relative spleen weight (11%), decreased relative thymus weight (12%), increased mean relative
brain weight (23-25%)
All other organ weight changes noted disappeared when when adjustment to decreasd body weight was performed.
Necropsy/Histopathology: No relevant macroscopic findings were noted in F1 and F2 pups. No details of microscopic
examinations were given in the publication.
Reproductive parameters: The paramete rs investigated (estrous cycle length, time to mating (precoital interval),
mating and fe rtility indices, gestation length) were unaffected during the F0 and F1 generations.
Litter parameters: Number of pups born per litter, mean live litter sizes, gender ratio, and postnatal survival were
unaffected by the test substance in the F0 and F1 generation.
Spermatogenic Endpoints: For none of the measured parameters in relation to sperm quality a toxicologically
significant difference was noted in the F0 and F1 generations.

Dose descriptor:

NOAEL

Remarks:

for developmental

Generation:

F1

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Reproductive effects observed:

not specified

None.

Conclusions:

In a 2-generation guideline study 30 rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate ad
libitum in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm. No adverse effe cts were found on
reproductive parameters such as estrous cyclicity, gonadal functions, spermatogenic endpoints (motility, morphology,
counts), mating behaviour and performance, conception, gestation and parturition, and fertility in general. No adverse
effects on the reproductive organs were noted.
Effe cts observed included unspecific systemic toxicity. In the high dose group mortality in F0 and F1 maternal animals
was observed, a decrease of mean body weights in m/f animals of F0 and F1 generation was noted (various time
points during the study) and an increase in relative liver weights (F0 and F1 females) found. In the mid dose group
effects observed included a slight decrease of mean body weights in F1 generation (various time points during the
study) and an increase in relative liver weights (F0 and F1 females). the increases in liver weights were not
accompanied by any macroscopic or microscopic findings. This holds also true for all other organs examined during
necropsy and histopatholgical evaluation. In the high and mid dose groups reduced postnatal pup weights were
observed for both sexes in the F1 and F2 generation.
As there were no effects on any of the measured parameters for effects on reproduction, the no -observed-adverseeffect
level (NOAEL) for reproductive toxicity was considered to be 10000 ppm.
Based on the described effects of general systemic toxicity at the mid and high dose group, the NOAEL for parental
toxicity was considered to be 3000 ppm.
The effe cts observed in the neonates (lower body and organ weights, both effects in the presence of maternal toxicity)
lead to the conclusion that the NOAEL fo r developmental toxicity can be established at 3000 ppm.

Executive summary:

In this Weight of Evidence appraoch, a well conducted GLP study by Faber at al (2007) on 2 -ethylhexylphthalate is used as

a read across to 2-ethylhexanol, one of the two hydrolytic byproducts of 00 -tert-amyl-0 -(2 -ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8), the registered substance.

Thirty Crl:CD®(SD)IGS BR rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm in this two generation reproduction toxicity study according to OECD guideline 416. Treatment of parental animals in the F0 generation started at 52 days of age and at postnatal day 22 (PND22) for the F1 generation. Animals received the diets for 70 consecutive days prior to mating, through the mating period (maximum of 14 days), during gestation and lactation until their scheduled necropsy (F0/F1 parental animals: 6 -8 days after weaning, F1 pups not selected for further generation and F2 pups at PND21). In guideline conform intervals cage side and detailed clinical observations, measurements of body weight and food consumption were performed. Various reproductive and developmental indices were assessed ( estrous cyclicity, gonadal functions, spermatogenic endpoints (motility, morphology, counts), mating behaviour and performance, conception, gestation and parturition, and fertility in general; total pup number/litter, mean live litter size, gender ratios, postnatal pup survival, developmental landmarks such as balanopreputial separation and vaginal patency). Parturition was allowed naturally as well as rearing of pups until weaning. On PND4 thirty F1 pups/sex/group were selected to constitute the F1 generation. Gross necropsies were performed on all animals. Histopathological examinations on various organs and tissues were performed on parental animals whereas for pups only organ weights were determined. Substance treatment had no adverse toxicological effect on any of the measured reproductive parameters for the F0 and F1 generations. Therefore the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was considered to be 10000 ppm.

In the high dose group deaths of 3 damns in the F1 generation and 7 damns in the F2 generation were attributed to substance treatment, as the deaths occurred 2 to 8 days after weaning - a period in which the damns maintain their body weights while still consuming large amounts of feed and thus resulting in high dose levels in the body (i.e. F0 = 745 mg/kg x d; F1 = 868 mg/kg x d). Single deaths in male animals were not treatment related. In high dose female animals of the F0 and F1 generation mean maternal body weights were reduced during gestation and lactation. Food consumption was also reduced in these dose groups during this time of exposure. In high dose group males of the F0 generation transient reductions of mean body weights occurred during various time points of the study. In the F1 generation males of the high and mid dose group showed reduced body weights starting from birth throughout the study. This was also true for females of the F1 in the mid dose group. Food consumption was reduced in males of the F1 generation in the high dose group throughout the study, whereas in the mid dose group this reduction was limited to the first week after weaning. In the high dose group increases in relative liver weights were observed in F0 and F1 females. This effect was not accompanied by any gross or microscopic findings and thus is not accounted for as adverse. Taken together these results indicate that a NOAEL for parental toxicity can be established at 3000 ppm.

In the high and mid dose group mean F1 and F2 male and female offspring weights were reduced on PND14 to 21. In the high dose group also reduced pup weights were observed at earlier time points (PND 1 and 7). In the high dose group in both sexes of F1 and F2 pups increased relative brain weights were found. In males of both generations decreased relative spleen weights were also observed. In female animals of the F2 generation reduced relative spleen and thymus weights were noted. In the mid dose group increased relative brain weights were found in females of the F1 generation. Based on these results, the NOAEL for developmental toxicity was considered to be 3000 ppm.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-01-11 to 2013-05-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: An apperently well conducted GLP study per information availablae in public domain on tert-amyl alcohol, one of the two hydrolytic byproducts of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8), the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

As described in the study report

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 9 - 10 weeks (supply), 10-11 weeks (start of pre-exposure), 10-11 weeks (start of exposure, day 0)
- Housing: Makrolon cages type M III, 1 animal, Exceptions: During mating: 1 male/1 female per cage, During rearing up to PND 4: 1 dam with her litter
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): humidity 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (06:00 -18:00 h), 12 hours darkness (18:00 - 06:00 h)

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

air

Details on exposure:

TEST SUBSTANCE PREPARATIONS AND ADMINISTRATION
For adaptation to the exposure conditions the animals were exposed to fresh air under comparable flow conditions in head-nose inhalation systems on several days before start of the exposure period.

GENERATION OF THE INHALATION ATMOSPHERE (VAPOR)
During exposure of the animals, exposure mixtures (inhalation atmospheres: test substance in air) were generated continuously in such a way that they are as homogeneous and as of a constant composition as possible.
Equipment:
- Piston metering pumps
- Two-component atomizers

Generation technique:
For each concentration, constant amounts of the substance to be tested were supplied to heated vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and passed into the inhalation systems.

EXPOSURE SYSTEMS
Head-nose inhalation systems:
The test atmospheres were passed into the aerodynamic exposure apparatuses (INA 60, V ≈ 90 L, BASF SE) with the supply air. The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamber to inhale the atmosphere.
The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air was lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones of the animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum period of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day [GD] 0)
- After successful mating each pregnant female was caged: If sperm are detected, mating of the pair will be discontinued.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measurement and recording of technical conditions in the exposure systems

In general, the technical parameters were measured and recorded as follows:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also be recorded by means of this system.
All these parameters were recorded continuously by an computerized data acquisition and control system.
The pump rate of the dosing pumps were read and recorded once per exposure. The atomizer pressure was measured continuously by manometers and recorded once per exposure.

Nominal concentration
The nominal concentration of the inhalation atmospheres was calculated from the amounts of test substance dosed and air-flow per unit time.

Analytical methods of determination

The constancy of concentrations in the inhalation atmospheres was surveyed continuously with total hydrocarbon analyzers (FID, Testa). As the measurements with FID presened the sum of the hydrocarbon in the air, to confirm the composition of the test atmosphere, the test atmospheres were analyzed once a week by gas chromatography of absorption samples.

Sampling and ananylses of absorption samples

Absorption samples were taken adjacent to the animals noses in order to confirm the identity of the test substance in the atmospheres. For this purpose, absorption vessels were connected in series, filled with appropriate solvent. Using a gas sampling station appropriate volume of atmosphere was drawn through the absorption vessels, which are analyzed by gas chromatography. Sampling frequency: one sample per concentration and week. The control atmosphere was sampled on one day during the exposure period.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

On 7 consecutive days per week for the desired period of time.

Details on study schedule:

Number of exposures:

Males:
a) 14 days premating
b) up to 14 days mating
c) Sacrifice after a minimum of 28 days after the first application

Females:
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19 d) after necropsy of the pups total 9 exposures on 9 consecutive days including the day before scheduled killing

Remarks:

Doses / Concentrations:
0,732, 2561,7316 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0,724, 2523, 6663 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

10 male and 10 female rats

Control animals:

yes

Details on study design:

- Dose selection rationale:
Based on available data, on approval by the sponsor, the following concentrations were selected for the present study:
7316 mg/m³ (2.000 ppm), as high concentration causing toxic effects
2561 mg/m³ (700 ppm), as mid concentration,
732 mg/m³ (200 ppm), as low concentration and expected NOAEC
(3.658 mg/m³ equates approximately 1ppm at room temperature and atmosheric pressure)

Test groups
Male and female Wistar rats were randomized according to their weight and allocated to the test groups before the beginning of the administration period. For each neurofunctional test and motor activity measurement, separate randomization lists were created (random selection).
Substitute animals were ordered with the animal supply, which was available for exchange until beginning of exposure. These animals were treated together with the study collective in the pre-exposure period. A health check of individual animals were performed on supply and on exchange.

EUTHANANSIA
- After the end of the administration period (at least 28 days) all surviving parental males were sacrificed and examined.
- The parental females were allowed to deliver and rear their pups until PND 4. On PND 4, all pups were sacrificed and examined as soon as possible.
- After PND 4 of the female, which delivered last, all parental females were exposed to the test substance on 9 consecutive days. They were sacrificed on the day after and were examined.

Parental animals: Observations and examinations:

MORTALITY: Yes
- Time schedule: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: On exposure days, a clinical inspection was performed on each animal at least three times a day (before, during and after exposure). On non-exposure days, a cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals were examined several times daily. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams were generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams were inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATION
All animals were subjected to detailed clinical observations (including palpation) outside their cages once before the administration period (day 0), and subsequently once per week (as a rule in the morning), by the same trained technicians, whenever possible. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal. The scope of examinations and the scoring of the findings that were observed were based on the current index of findings in Tox-Lims software and includes but was not limited to the following parameters listed:

1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parental animals were determined once a week at the same time of the day (in the morning), if possible. The following exceptions were notable for the female parental animals:
- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as the males. Body weight data was only reported in the individual tables.
- Females with litter were weighed on the day after parturition (PND1) and on PND 4.
- Females without litter were weighed once a week. The body weight data of these individuals were only reported in the individual tables.
- After weaning (PND 4), females were weighed once a week until sacrifice; body weight data were only reported in the individual tables.
- After the pups were sacrificed the females were exposed for 9 consecutive days. The F0 females were weight once on the first exposure of this exposure period, once on the third and once on the eight exposure. The last body weight determination was on the day of the gross necropsy.


FOOD CONSUMPTION
Generally, food consumption was determined once a week for the male and female parental animals.
- Food consumption was determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4.
- Food consumption of the females during the 9 exposure days after necropsy of the pups was determined over one week from study day 47 to day 54.

Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

FUNCTIONAL OBSERVATION BATTERY (FOB)
The FOB was carried out in 5 surviving parental males and females per group. For the males the FOB was carried out at the end of the administration period, for the females at the end of the premating period. No inhalation exposure took place on the day of FOB examination for all animals of the respective sex.

Home cage observation
The rats were observed for about 10-30 seconds in their closed home cages. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. Other findings

Open field observation
The rats were transferred to a standard arena (50 x 50 cm with sides of 25 cm height) and observed for at least 2 minutes. In addition to abnormalities, the following parameters were examined with particular attention:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalites
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearings within 2 minutes

Sensory-motoric test/Reflexes
The rats were then removed from the open field and subjected to the following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

MOTOR ACTIVITY MEASUREMENT
The MA was carried out in 5 surviving parental males and females per group. For the males the MA was carried out at the end of the administration period, for the females at the end of the premating period.
The MA was carried out on the same day as the FOB was performed. The examinations were performed using the TSE-Labmaster System, TSE , Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Four beams were allocated per cage. The number of beam interrupted was counted over 12 intervals for 5 minutes per interval.

CLINICAL PATHOLOGY
Blood samples were taken from fasted animals by puncturing the retrobulbar venous plexus under Isoflurane anesthesia. Blood sampling and examination were carried out in a randomized sequence.
The parameters listed below were examined in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

- Hematology
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Preparation of blood smears (only evaluated blood smears will be archived)
12. Prothrombine time

- Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum γ-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. Inorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol
19. Bile acid

- Hormones
Additional serum samples were frozen at -80°C for storage. Measurement of T3, T4 and TSH were carried out only if there was an indication for an effect on pituitary-thyroid axis. The determination was triggered based upon alterations of thyroid histopathology. Depending on the results obtained, samples which were not examined, were stored not longer than 1 year after finalization of the report.

Litter observations:

PUP STATUS AND LITTER SIZE AFTER BIRTH
Status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were also examined for gross-morphological changes.

PUP VIABILITY/MORTALITY
In general, a check was made for any dead or moribund pups twice daily on workdays or as a rule, only in the morning on Saturdays, Sundays and public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

CLINICAL SIGNS
All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented for each pup.

BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4. The body weight determined on PND 1 was also used to determined runts. Those pups whose body weight was 25% below the mean body weight of the control group (separately according to male and female pups) were defined as runts.

Postmortem examinations (parental animals):

NECROPSY
The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. Animals which died intercurrently or were killed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Lung
7. Spleen
8. Thymus

ORGAN/TISSUE FIXATION
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (tracheobronchial, mediastinal and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladde
48. Uterus
49. Vagina
HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the list below:
1. Adrenal glands
2. All gross lesions
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Larynx (3 levels)
17. Lungs (5 lobes)
18. Lymph nodes (tracheobronchial, mediastinal)
19. Lymph nodes (mesenteric)
20. Nasal cavity (4 levels)
21. Ovaries
22. Oviducts
23. Prostate gland
24. Peyer’s patches
25. Rectum
26. Sciatic nerve
27. Seminal vesicles
28. Spinal cord (cervical, thoracic, lumbar)
29. Spleen
30. Stomach (forestomach and glandular stomach)
31. Testes
32. Thymus
33. Thyroid glands
34. Trachea
35. Urinary bladder
36. Uterus
37. Vagina

Special attention was given on stages of spermatogenesis in the male gonads. Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals wer prepared if required.

Postmortem examinations (offspring):

POSTMORTEM EXAMINATIONS OF PUPS
On PND 4, the pups were sacrificed under isoflurane anesthesia with CO2. After sacrificed pups were examined externally and eviscerated, and their organs were assessed macroscopically. Pups that die or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death. All pups without any notable findings were discarded after their macroscopic evaluation.

Reproductive indices:

The testes, epididymides and ovaries of animals that died or had to be sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites.

Offspring viability indices:

Yes

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive performance:

no effects observed

In males and females of test group 3 (7316 mg/m3), several clinical signs (unconsciousness, apathy, piloerection, reduced attention and unsteady gaits) were observed (during and after exposure) indicating narcotic effect of the test substance. In addition, animals showed some unspecific signs as alopecia and reduced fur care indicating bad general condition of the animals. Occasionally, gasping was observed in single animals.Moreover, reduced attention, apathy, reduced fur care, blood in bedding and vaginal discharge were observed in females of test group 3.

Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls.

The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3) and 3. The mean body weights of the test group 3 male and female animals were significantly lower than the control group.

The mating index was 100% in all groups including the controls. The fertility index varied between 80 and 100 % and reflect the normal range of biological variation inherent in the strain of rats used for this study. The mean number of implantation sites, post implantation loss and liveborn pups was comparable between all test substance-treated groups and the controls.

Regarding histopathology, female animals of test group 3 revealed a reduction of vaginal epithelial height and hypertrophy with increase of mucification. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEC

Remarks:

Reproduction toxicity

Effect level:

2 561 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Effect level:

2 561 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Generation: General toxicity (migrated information)

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

No effects were observed regarding pup viability, sex ratio and pup body weights.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

7 316 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Reproductive effects observed:

not specified

Conclusions:

The Weight of Evidence approach was used to examine the reproductive toxicity of 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate.. NOAEC for reproduction, parental (general) and F1 toxicities were determined to be 2561, 2561, and 7316 mg/m3, respectively, for t-amyl alcohol (CAS No. 75 -85 -4), one of the byproducts of hydrolysis of the test item, 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8).

Executive summary:

The registered substance, 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8) is believed to be hydrolyzed into two major hydrolysis byproducts, 2-ethylhexanol (CAS No.104-76-7) and t-amyl alcohol (CAS No. 75-85-4). t-amyl alcohol may be further oxidized to diols and carboxylic acids and these reactions may be mediated by cytochrome p450-dependent oxidations. Therefore, available reproductive toxicological data from t-amyl alcohol were used as the Weight of Evidence (WOE) approach to satisfy the reproductive and developmental toxicity end point data requirements of 00 -tert-amyl-0 -(2 -ethylhexyl)monoperoxycarbonate.

A combined repeated dose and reproductive/developmenatal toxicity screening test (OECD 422) was done under GLP conditions in male and female Wistar rats. The test article, t-amyl alcohol (CAS No. 75-85-4) was administered by inhalation (vapor) at nominal concentrations of 0, 732, 2561, and 7316 mg/m3 for test periods (premating, mating, pregancy) in accordance with the OECD 422 guidelines.

Several clinical signs in males and females of test group 3 (7316 mg/m3) and blood in bedding and vaginal discharge were observed in females of test group 3. Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls. The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3) and 3. The mean body weights of the test group 3 male and female animals were significantly lower than the control group. Regarding histopathology, female animals of test group 3 revealed a reduction of vaginal epithelial height and hypertrophy with increase of mucification.

The mating index was 100% in all groups including the controls. The fertility index varied between 80 and 100 % and reflect the normal range of biological variation inherent in the strain of rats used for this study. The mean number of implantation sites, post implantation loss and liveborn pups was comparable between all test substance-treated groups and the controls. No effects were observed regarding pup viability, sex ratio and pup body weights.

NOAEC for reproduction, parental (general) and F1 toxicities were determined to be 2561, 2561, and 7316 mg/m3, respectively, for t-amyl alcohol (CAS No. 75 -85 -4), one of the byproducts of hydrolysis of the registered item, 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8).

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 20 September 2017 Experimental completion date: 22 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate
Test item identity (including alternative names): tert-Amylperoxy-2-ethylhexylcarbonate
Trigonox 131
CAS number: 70833-40-8
Intended use: Industrial chemical
Appearance: Clear colorless liquid
Storage conditions: Frozen (-10 to -30C) in the dark
Supplier: Sponsor
Batch number: 1702442114
Retest date: 24 March 2019
Purity: 96.5%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS (UK) Limited.
Number of animals ordered 44 males and 48 females (92 in total; included 4 spare males and 8 spare females)
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment Males: 84 to 90 days old.
Females: 98 to 104 days old.

Weight range of the animals at the start of treatment Males: 319 to 358 g.
Females: 199 to 227 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.

Groups were adjusted to reduce inter-/intra-group variation.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Animal Care and Husbandry
Environmental Control

Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing for mating; these were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) and discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Route of administration:

oral: gavage

Vehicle:

other: PEG300

Details on exposure:

Correction factor
Used as supplied; no correction factor applied.

Vehicle PEG300.

Method of preparation
O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was prepared for administration as a series of graded concentrations in the vehicle. Starting with the low concentration (50 mg/mL), the formulation was prepared by weighing out the required amount of test item, adding 40 to 50% of the final volume of vehicle and magnetically stirring until all the test item was uniformly mixed. The solution was made up to the required volume with the vehicle and stirred using a magnetic stirrer until homogenous.

The procedure was repeated for the mid and high dose (50 and 200 mg/mL).

Frequency of preparation
Weekly.

Storage of formulation
Refrigerated (2-8ºC).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Details on mating procedure:

Mating Procedure
Paired for mating After two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis

Validated concentration range
20 mg/mL to 200 mg/mL
GLP Study: Harlan Laboratories Study Report: D45837 (homogeneity and stability establish for 4 hours at ambient temperature and 8 days refrigerated storage); 10 mg/mL validated as part of this study.
Stability assessment Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at a concentration of 10 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration during the first and last week of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in acetonitrile (50 mL).

Solutions for instrument calibration were prepared by appropriate dilution of the primarystandard using diluent and contained O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at nominal concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL and 250 μg/mL.

Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurate volume or weight where applicable) was dissolved in a suitable volume of acetonitrile. The extract was diluted using diluent to provide a solution containing O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at an expected concentration within the range 100 μg/mL to 200 μg/mL.

The concentration of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in the final solution was quantified by HPLC using UV detection.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (PEG300) with known amounts of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: XBridge Shield RP18, 75 × 4.6 mm, 3.5 μm
Column temperature: 45ºC
Sample temperature: 4ºC
Mobile Phase: ACN/water/ o-H3PO4 65/35/0.1 v/v/v
Flow rate: 1 mL/min
Needle wash: ACN/water 50/50 v/v
Detector wavelength: UV, 210 nm
Injection volume: 20 μL
Run time: 8 minutes
Approximate retention time: 4.3 minutes

Duration of treatment / exposure:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation

Frequency of treatment:

Daily

Details on study schedule:

Study initiation (Study Plan signed by Study Director) 21 August 2017

Experimental start date
(Pre-Study chemistry) 20 September 2017
Animal arrival
Males 4 october 2017
Females 20 September 2017

Estrous cycle evaluation commenced 26 September 2017

Treatment commenced 10 October 2017

F0 pairing commenced 24 October 2017

Necropsy
F0 Males 7 November 2017
F0 Females/F1 Offspring 28 November to 2 December 2017

Experimental completion date 22 March 2018

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males
10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Dose levels of 50, 250 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from an OECD 407 study conducted at 100, 300 and 1000 mg/kg/day (Harlan Laboratories Study Report D45837). In this study findings included:
• Reductions in food consumption at 1000 mg/kg/day
• Slightly high liver weights for females at 300 or 1000 mg/kg/day, with two animals exhibiting minimal centrilobular hepatocellular hypertrophy; considered to be metabolic adaption and therefore not adverse.
• Increased severity of hyaline droplets in the renal proximal tubules of males at 1000 mg/kg/day; this is considered to be an adverse finding in rats but has no toxicological significance in man.
• Forestomach mucosal necrosis for males at 100 mg/kg/day and for both sexes at 300 mg/kg/day or 1000 mg/kg/day; these were considered to be local injuries that resulted in subsequent adverse reactions.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for systemic toxicity was 1000 mg/kg/day. However the no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach.

On this study a high dose of 1000 mg/kg/day was anticipated to elicit parental effects in terms of food consumption, liver pathology and stomach lesions. A low dose of 50 mg/kg/day was selected with the aim to establish a NOEL, with an intermediate dose of 250 mg/kg/day to aid interpretation of any dose response.

Positive control:

Not applicable

Parental animals: Observations and examinations:

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily
Week 2 onwards - once each week

F0 females
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose
One to two hours after completion of dosing
As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males
Once each week

F0 females
Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13

Body Weight
The weight of animals was recorded as follows:
F0 males
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before mating.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals
Weekly, before pairing.

For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Oestrous cyclicity (parental animals):

Estrous Cycles
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:

For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.

For four days before scheduled termination (nominally Days 10-13 of lactation).

Sperm parameters (parental animals):

Testes weight and histopathology

Litter observations:

Records Made During Littering Phase
Clinical observations
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all offspring.
Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Tissue and regions examined
Abnormalities
Epididymides (caput, corpus and cauda)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulation gland)
Stomach
Testes
Thyroid
Uterus with cervix and oviducts

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.
For apparently empty uterine horns The absence of uterine implantation sites was confirmed by
staining with ammonium sulphide [modification of the
Salewski staining technique (Salewski, E, 1964)].

Female whose litter died before Day 13 of lactation Mammary tissue appearance.

Light Microscopy
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

Thyroid hormone analysis
- blood samples were collected as follows

Day 4 of age
F1 offspring, two females per litter (where possible).
• one for T4 (serum)#
• one for TSH (plasma)

No females were allocated to these procedures if:
• The resultant live litter size fell below eight offspring.
• The resultant number of live females fell to less than three

If only four female offspring were available in a litter but the overall litter size was more than eight, one female pup was selected with priority given to the serum sample.
# priority was given to serum sample

Day 13 of age
F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample


Premature deaths
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

F1 offspring on Day 13 of age
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormal offspring were retained in appropriate fixative.
Thyroid glands were preserved from two offspring - one male and one female in each litter, where possible.

Statistics:

Please refer to "Any other information on materials and methods, including tables"

Reproductive indices:

Mating Performance and Fertility

Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to administration of the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight change for males during the four week treatment period was slightly low for males receiving 1000 mg/kg/day at approximately 83% of Controls; this difference did not attain statistical significance. Body weight change at 50 and 250 mg/kg/day was unaffected by treatment.

Body weight change for females during Week 1 of treatment was slightly low at 250 and 1000 mg/kg/day at approximately 57% of Controls; however this difference did not attain statistical significance. The overall weight gain for females for the two weeks before pairing, during gestation and lactation were unaffected by treatment at dose levels up to and including 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption for males for the two weeks before pairing was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.
During the first week of treatment females at 1000 mg/kg/day had slightly but not statistically significant low food consumption, approximately 89% of Controls. At 50 and 250 mg/kg/day food consumption was similar to Controls.

During gestation food consumption was considered unaffected by treatment.

During lactation the overall food consumption for females receiving 1000 mg/kg/day was marginally low when compared with Controls (approximately 93% of Controls), however the mean phase values were not statistically different from Controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related findings in the stomach of three males and one female that received 250 mg/kg/day and eight males and one female that received 1000 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The number of implantations and subsequently the live litter size on Days 1 and 4 of age (before selection of offspring for thyroid hormone sampling) were statistically significantly low at 1000 mg/kg/day when compared with concurrent Controls (implantations - p<0.01; litter size - p<0.05) and review of the historical control data showed that two litters on Day 4 were outside the 90 percentile range (7.7-14.4; n=74). Litter size at 50 or 250 mg/kg/day was unaffected by parental treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Implantation count

Key result

Dose descriptor:

NOEL

Remarks:

Parental effects

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Parental effects

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects noted on overall parental effcts

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

There no signs at routine physical examination of the offspring that could be attributed to parental treatment

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Offspring survival was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.

At 1000 mg/kg/day one litter comprising of 13 offspring was found dead prior to Day 1 of age (litter no 140); the offspring weighed between 4.4 and 5.3 g. Macroscopic examination of the dam (no. 140) revealed 13 uterine implantation sites and pale/inactive mammary tissue. In the absence of any effect on offspring survival in the remaining litters this isolated litter death is considered to be unrelated to parental treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring bodyweight on Day 1 of age and subsequent weight gain up to Day 4 of age was considered to be unaffected by parental treatment.
At 1000 mg/kg/day body weight gain for male offspring from Day 4 to 7 of age was statistically significantly low when compared with Controls (p<0.05; approximately 83% of Controls). Female offspring at the same dose level showed marginally low weight gain over the same period (91 % of Controls), however this difference did not attain statistical significance. Offspring body weight gain from Day 7 to Day 13 of age and the overall weight gain from Day 1 to Day 13 of age was similar across the groups. Therefore this slight effect on offspring bodyweight gain at 1000 mg/kg/day was not considered to be adverse
Offspring body weight gain at 50 and 250 mg/kg/day was unaffected by parental treatment.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that died prematurely and those at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to parental treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

Litter size, sex ratio and survival indices
At 1000 mg/kg/day one litter comprising of 13 offspring was found dead prior to Day 1 of age (litter no 140); the offspring weighed between 4.4 and 5.3 g. Macroscopic examination of the dam (no. 140) revealed 13 uterine implantation sites and pale/inactive mammary tissue. In the absence of any effect on offspring survival in the remaining litters this isolated litter death is considered to be unrelated to parental treatment.

Sex ratio was unaffected by parental treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Offspring development

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

It was concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. 

Conclusions:

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.

Executive summary:

  Summary

This study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with the administration of the test item [O‑(2‑ethylhexyl) O,O-tert-pentyl peroxycarbonate], an industrial chemical, by oral gavage, to Sprague Dawley rats for at least four weeks.

Three groups of ten male and ten female rats received test item at doses of 50, 250 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, PEG300, at the same volume dose as treated groups.

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results and Conclusion

Treatment ofO-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to parental animals at 50, 250 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance or fertility. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

Treatment related findings were restricted to:

·        Low implantations and subsequent live litter size on Days 1 and 4 of age at 1000 mg/kg/day. This effect was slight and in isolation with no effects on any other reproductive parameters or micropathology of the reproductive organs.

·        Macroscopic and microscopic changes in the stomach of parental animals at 250 and 1000 mg/kg/day. The microscopic findings of epithelial hyperplasia and submucosal infiltration of inflammatory cells in the non-glandular region of the stomach were considered to be indicative of a local irritant effect of the test item at 250 and 1000 mg/kg/day, with a dose-relationship in male animals; in males this correlated with gross findings of a thickened forestomach.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.

Reference 4

Endpoint:

toxicity to reproduction

Remarks:

other: Bichemical or cellular interactions on testes / sertoli cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable publication which meets basic scientific principles. This study is on 2-ethylhexanol, one of the two hydrolytic byproducts of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8), the registered substance.

Qualifier:

no guideline available

Principles of method if other than guideline:

Biochemical or cellular interactions effect on testes / sertoli cells

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

As described in the TMI

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: propylene glycol (MEHP and MEHP-metabolites; DEHP and 2-EH: suspension without vehicle

Details on exposure:

5 exposures

Frequency of treatment:

1/day

Remarks:

Doses / Concentrations:
2.7 mmol/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

6 animals per test substance

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

In vivo: metabolite blood levels (excluding 2-EH). Absolute and relative testes and prostate weight; degenerate dcells
in seminiferous tubules.
In vitro: germ cell detachment in primary rat testicular cell cultures.

Clinical signs:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Degeneration of testes and Sertoli cells were not associated with 2 -EH, but with MEHP, which is generated following the
administration of DEHP.

Dose descriptor:

NOAEL

Effect level:

2.7 other: mmol/kg/d

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No testicular damage observed when 2.7 mmol/kg/day was administered to rats

Dose descriptor:

NOAEL

Effect level:

200 other: micromolar

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No germ cell detachment seen in testicular culture when the test substance was added in vitro upto 200 micromolar concentration

Remarks on result:

other: Generation not specified (migrated information)

Clinical signs:

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

not applicable

Remarks on result:

other: not applicable

Reproductive effects observed:

not specified

No other information

Conclusions:

MEHP is the metabolite that is responsible for the testicular damage that is observed following administration of DEHP
to rats. 2-EH has no adverse effect on testis, germ cell detachment in vivo or in vitro.

Executive summary:

In was shown vivo and also in primary rat testicular cell cultures in vitro that MEHP was the metabolite that causes testicular damage following administration of DEHP. MEHP caused a significantly increased germ cell detachment in vitro at 1 μM and above, whereas effcts of 200 μM DEHP and 2 -EH were not different from controls after 24 or 48 hour incubation in vitro. 2-EH has no adverse effect on testis, germ cell detachment in vivo or in vitro (Sjöberg et al., 1986). Taken together, these results provide supportive evidence that 2 -ethylhexanol, one of the hydrolytic byproducts of the registered substance does not show toxicity to the male reproductive system at the dose tested and under the conditions tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good (GLP study)

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 561 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

An OECD 422 study (rats, inhalation exposure) conducted per GLP regulations on t-amyl alcohol, one of the hydrolytic products of the registered substance gave the above results.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The OECD 421 study of the test substance in orally gavaged rats resulted in an NOAEL of 1000 mg/kg bw/d for systemic effects and 50 mg/kg bw/d for local effects (stomach irritation) in parental animals and an NOAEL of 250 mg/kg bw/d for reproductive toxicity based on reproductive performance and offspring development.

The above registered substance, O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8) is normally hydrolyzed to two major byproducts, 2-ethylhexanol (CAS No.104-76-7) and t-amyl alcohol (CAS No. 75-85-4). Therefore, available reproductive toxicological data on t-amyl alcohol and 2 -ethylhexanol was used as the Weight of Evidence (WOE) approach to support the reproductive toxicity end point of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate. No reproductive toxicity of t-amyl alcohol was observed under the conditions of the study (NOAEC: 2561 mg/m3).

Effects on developmental toxicity

Description of key information

In an oral gavage study in rats (OECD 421; Reproductive/developmental screening test), the test item did not cause any toxicologically significant developmental effect at or below 250 mg/kg bw/d, thus providing the latter as the NOAEL value (Envigo study, 2018).

Supportive evidence from reprodcutive/developmental studies on the registered substance's breakdown products are also prsented.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An apperently well conducted GLP study per information availablae in public domain on 2-ethylhexanol, one of the two hydrolytic byproducts of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8), the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

As described in the TMI

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

- CD-1 Swiss mice
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC, USA
- Weight at study initiation: range 23.52-31.59 g
- Housing: individually in solid-bottom polycarbonate cage swith stainle ss steel wire lids
- Diet: ad libitum
- Water: ad lib itum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature: 72°F
- Humidity (%): 48
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: microencapsulation

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): fed was prepared once. Fresh supplies of dosed feed were obtained from
refrigera ted s tock e very 3 days.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodnet Chow
- Storage temperature of food: refrigerated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of 2 -EH in the feed was analyzed by gas chromatography (GC) prior to use.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Gestational days 0 to 17

Frequency of treatment:

7/week

Duration of test:

17 days

Remarks:

Doses / Concentrations:
0, 17, 59, 191 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

Microencapsulated 2-EH (0%, 0.009%, 0.03%, or 0.09% in fe ed) was provided on gestational days (gd) 0 to 17 ad libitum to tim ed-mated CD-1® mice
(28/group).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Tim e schedule: daily
- Cage side observations checked in table [Appendis I-11] were included.
BODY WEIGHT: Yes
- Time schedule for examinations: gestational day 0, 3, 6. 9. 12, 15, 17
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/k g body
weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 17
- Organs examined: liver and uterus

Ovaries and uterine content:

At sacrifice (gd 17), the number of ovarian corpora lutea and uterine implantation sites, including resorptions, and
dead or live fetuses, were recorded.

Fetal examinations:

Live and dead fetuses were weighed. Live fetuses were sexed and examined for external, visceral and skeletal
malformations and varia tions.

Statistics:

General Linear Models (GLM) procedures were applied fo r the analysis of variance (ANOVA) of maternal and fetal
parameters. Bartlett's test for homogeneity of variance was performed an all data to be analyzed by ANOVA. When
ANOVA revealed a significant (p<0.05) dose effect, Dunnett’s Multiple Comparison Test was used to compare each o f
the treated groups with the control groups. Other analyses comprised chi square test and Fisher’s exact probability
test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No dams died, delivered early or were removed from study. P regnancy rate was high (93 -96%) and equivalent across
all groups. One litter at 0%
was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters
evaluated were 27 at 0.009 and
0.03% and 26 at 0 and 0.09% 2-EH.
There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains
(absolute or corrected for gra vid
uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food
consumption (g/kg/day and g/day) was significantly increased for gd 0 -3 a t 0.09% and unaffected fo r all other time
points evaluated. The calculated consumption of 2 -EH, based on gestational food consumption was 0 (0 mmol/kg), 17
(0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups,
respectively.

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effe cts of exposure to dietary 2-EH on any gestational parameters. The number of corpora lutea,
uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio (%, males) and live fetal
body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no
tre atment-rela ted changes in the incidence of individual, external, visceral, sk eletal or total m alformations or
variations.

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

No dams died, delivered early or were removed from study. Pregnancy rate was high (93-96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and 26 at 0 and 0.09% 2-EH. There was no treatmentrelated maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for gestational das 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0 mmol/kg), 17 (0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively. There were no effects of exposure to dietary 2-EH on any gestational parameter. The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio (%, males) and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatmentrelated changes in the incidence of individual, external, visceral, skeletal or total malformations or variations.

Conclusions:

No maternal or developmental toxicity was observed in a mouse oral feed study at (equivalent to OECD TG 414,
dosing during gestation days 0-17 at any dose up to and including 191 mg/kg bw/day, the highest tested dose.

Executive summary:

In this Weight of Evidence appraoch, a well conducted GLP study by Tyl et al (1991) on 2 -ethylhexanol, one of the hydrolytic

byproducts of 00 -tert-amyl-0 -(2 -ethylhexyl)monoperoxycarbonate (CAS No. 70833 -40 -8), the registered substance is used.

2-EH was examined in this mouse feed study for its potential for developmental toxicity equivalent to OECD TG 414 and under GLP conditions. Timed pregnant female CD-1 Swiss mice (28 animals/group, body weight range 32.5 to 31.6 g) received 2-EH in the diet at nominal concentrations of 0, 0.009, 0.03, and 0.09% during gestation days 0-17. The animals were housed singly and observations for clinical signs were made daily. Body weights were recorded on gestational day 0, 3, 6, 9, 12, 15, 17. Food consumption and test compound intake were calculated individually. Test substance purity and concentration in the diets was verified using gas chromatography. Test substance purity was >99%. C oncentration in the diets was within 99-108% of the nominal concentration.

Maternal effects: Food intake and hence dose levels were higher than expected. Average intakes were 0, 17, 59, and 191 mg/kg bw/day, respectively. No dams died, delivered early or were removed from study. Pregnancy rates were high (93- 96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and 26 at 0 and 0.09% 2- EH.

There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for GD 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0 mmol /kg), 17 (0.13 mmol /kg), 59 (0.46 mmol /kg) and 191 mg/kg/day (1.49 mmol /kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

Fetal effects: There were no effects of exposure to dietary 2-EH on any gestational parameters.The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations.

In conclusion, there were no maternal or developmental toxic effects of 2-EH dietary exposure throughout gestation at any concentration tested. The NOAEL for maternal toxicity and for developmental toxicity and teratogenicity was therefore 191 mg/kg bw/day, the highest dose level tested. This study was conducted equivalent to OECD TG 414 and under GLP conditions, and it is considered to be valid without restriction ( Tyl et al., 1991).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-01-11 to 2013-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An apperently well conducted GLP study per information availablae in public domain on tert-amyl alcohol, one of the two hydrolytic byproducts of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8), the registered substance.

Qualifier:

according to guideline

Guideline:

other: OECD guideline 422 [Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OTP 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

As described in the TMI

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633
Sulzfeld
- Age at study initiation: 9 - 10 weeks (supply), 10-11 weeks (start of pre-exposure), 10-11 weeks (start of exposure,
day 0)
- Housing: Makrolon cages type M III, 1 animal, Exceptions: During mating: 1 male/1 female per cage, During rearing
up to PND 4: 1 dam with her litter
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): humidity 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (06:00 -18:00 h), 12 hours darkness (18:00 - 06:00 h)

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

air

Details on exposure:

TEST SUBSTANCE PREPARATIONS AND ADMINISTRATION
For adaptation to the exposure conditions the animals were exposed to fresh air under comparable flow conditions in
head-nose inhalation systems on several days before start of the exposure period.
GENERATION OF THE INHALATION ATMOSPHERE (VAPOR)
During exposure of the animals, exposure mixtures (inhalation atmospheres: test substance in air) were generated
continuously in such a way that they are as homogeneous and as of constant composition as possible.
Equipment:
- Piston metering pumps
- Two-component atomizers
Generation technique:
For each concentration, constant amounts of the substance to be tested were supplied to heated vaporizers by means
of metering pumps. The vapors were mixed with streams of conditioned air and passed into the inhalation systems.
EXPOSURE SYSTEMS
Head-nose inhalation systems:
The test atmospheres were passed into the aerodynamic exposure apparatuses (INA 60, V ≈ 90 L, BASF SE) with the
supply air. The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamber to inhale
the atmosphere.
The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air
was lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the
breathing zones of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measurement and recording of technical conditions in the exposure systems

In general, the technical parameters were measured and recorded as follows:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also be recorded by means of this system.

All these parameters were recorded continuously by an computerized data acquisition and control system. The pump rate of the dosing pumps were read and recorded once per exposure. The atomizer pressure was measured continuously by manometers and recorded once per exposure.

Nominal concentration
The nominal concentration of the inhalation atmospheres was calculated from the amounts of test substance dosed and air-flow per unit time.

Analytical methods of determination
The constancy of concentrations in the inhalation atmospheres was surveyed continuously with total hydrocarbon analyzers (FID, Testa). As the measurements with FID presened the sum of the hydrocarbon in the air, to confirm the composition of the test atmosphere, the test atmospheres were analyzed once a week by gas chromatography of absorption samples.

Sampling and ananylses of absorption samples
Absorption samples were taken adjacent to the animals noses in order to confirm the identity of the test substance in the atmospheres (sampling velocity in the sampling probe = 1.25 m/sec). For this purpose, absorption vessels were connected in series, filled with appropriate solvent. Using a gas sampling station appropriate volume of atmosphere was drawn through the absorption vessels, which are analyzed by gas chromatography. Sampling frequency: one sample per concentration and week. The control atmosphere was sampled on one day during the exposure period.

Details on mating procedure:

M/F ratio per cage: 1:1
- Length of cohabitation: maximum period of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day [GD] 0)
- After successful mating each pregnant female was caged: If sperm are detected, mating of the pair will be
discontinued

Duration of treatment / exposure:

6 hours

Frequency of treatment:

On 7 consecutive days per week for the desired period of time

Remarks:

Doses / Concentrations:
0,732, 2561,7316 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0,724, 2523, 6663 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

10 male and 10 female rats

Control animals:

yes

Details on study design:

- Dose selection rationale:

Based on available data, on approval by the sponsor, the following concentrations were selected for the present study:
7316 mg/m³ (2.000 ppm), as high concentration causing toxic effects
2561 mg/m³ (700 ppm), as mid concentration,
732 mg/m³ (200 ppm), as low concentration and expected NOAEC
(3.658 mg/m³ equates approximately 1ppm at room temperature and atmosheric pressure)

Test groups
Male and female Wistar rats were randomized according to their weight and allocated to the test groups before the beginning of the administration period. For each neurofunctional test and motor activity measurement, separate randomization lists were created (random selection). Substitute animals were ordered with the animal supply, which was available for exchange until beginning of exposure. These animals were treated together with the study collective in the pre-exposure period. A health check of individual animals were performed on supply and on exchange.

EUTHANANSIA
- After the end of the administration period (at least 28 days) all surviving parental males were sacrificed and examined.
- The parental females were allowed to deliver and rear their pups until PND 4. On PND 4, all pups were sacrificed and examined as soon as possible.
- After PND 4 of the female, which delivered last, all parental females were exposed to the test substance on 9 consecutive days. They were sacrificed on the day after and were examined.

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.
CLINICAL OBSERVATIONS: Yes
- Time schedule: On exposure days, a clinical inspection was performed on each animal at least three times a day
(before, during and after exposure). On non-exposure days, a cageside examination was conducted at least once
daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the
animals were examined several times daily. Abnormalities and changes were documented for each animal. The
parturition and lactation behavior of the dams were generally evaluated in the morning in combination with the daily
clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were
documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition
behavior of the dams were inspected in the afternoons in addition to the evaluations in the mornings.
DETAILED CLINICAL OBSERVATION
All animals were subjected to detailed clinical observations (including palpation) outside their cages once before the
administration period (day 0), and subsequently once per week (as a rule in the morning), by the same trained
technicians, whenever possible. For observation, the animals were removed from their cages and placed in a standard
arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal. The scope of examinations and
the scoring of the findings that were observed were based on the current index of findings in Tox -Lims software and
includes but was not limited to the following parameters listed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parenta l animals were determined once a week at the same time
of the day (in the morning), if possible. The following exceptions were notable for the female parental animals:
- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7,
14 and 20.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating
interval as the males. Body weight data was only reported in the individual tables.
- Females with litter were weighed on the day after parturition (PND1) and on PND 4.
- Females without litter were weighed once a week. The body weight data of these individuals were only reported in the
individual tables.
- After weaning (PND 4), females were weighed once a week until sacrifice; body weight data were only reported in the
individual tables.
- After the pups were sacrificed the females were exposed for 9 consecutive days. The F0 females were weight once on
the first exposure of this exposure period, once on the third and once on the eight exposure. The last body weight
determination was on the day of the gross necropsy.
FOOD CONSUMPTION
Generally, food consumption was determined once a week for the male and female parental animals.
- Food consumption was determined after the 2nd premating week (male parental animals) and during the mating
period (male and female parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4.
- Food consumption of the females during the 9 exposure days after necropsy of the pups was determined over one
week from study day 47 to day 54.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation
periods and in the females without litter during lactation period.

FUNCTIONAL OBSERVATION BATTERY (FOB)
The FOB was carried out in 5 surviving parental m ale s and females per group. For the males the FOB was carried out
at the end of the administration period, for the females at the end of the premating period. No inhalation exposure
took place on the day of FOB examination for all animals of the respective sex.
Home cage observation
The rats were observed for about 10-30 seconds in their closed home cages. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. Other findings
Open field observation
The rats were transferred to a standard arena (50 x 50 cm with sides of 25 cm height) and observed for at least 2
minutes. In addition to abnormalities, the following parameters were examined with particular attention:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalites
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearings within 2 minutes
Sensory-motoric test/Reflexes
The rats were then removed from the open field and subjected to the following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupilla ry reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings
MOTOR ACTIVITY MEASUREMENT
The MA was carried out in 5 surviving parental males and females per group. For the males the MA was carried out at
the end of the administration period, for the females at the end of the premating period.
The MA was carried out on the same day as the FOB was performed. The examinations were performed using the TSELabmaster
System, TSE , Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages
with a small amount of bedding for the duration of the measurement. Four beams were allocated per cage. The
number of beam interrupted was counted over 12 intervals for 5 minutes per interval.
CLINICAL PATHOLOGY
Blood samples were taken from fasted animals by puncturing the retrobulbar venous plexus under Isoflurane
anesthesia. Blood sampling and examination were carried out in a randomized sequence.
The parameters listed below were examined in the first 5 surviving parental male s and the first 5 surviving females
with litter (in order of delivery) per group.
- Hematology
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Preparation of blood smears (only evaluated blood smears will be archived)
12. Prothrombine time
- Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum γ -glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. Inorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol
19. Bile acid
- Hormones
Additional serum samples were frozen at -80°C for storage. Measurement of T3, T4 and TSH were carried out only if
there was an indication for an effect on pituitary-thyroid axis. The determination was triggered based upon alterations
of thyroid histopathology. Depending on the results obtained, samples which were not examined, were stored not
longer than 1 year after finalization of the report.
NECROPSY
The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena
cava. The animals were necropsied and assessed by gross pathology. Anim als which died intercurrently or were killed
in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.
ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule :
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Lung
7. Spleen
8. Thymus
ORGAN/TISSUE FIXATION
The following organs or tissues of a ll parental animals were fixed in 4% buffered formaldehyde solution or modified
Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (tracheobronchia l, mediastinal and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skele tal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladde
48. Uterus
49. Vagina
HISTOPATHOLOGY
Fixation was fo llowed by histotechnical processing, examination by light microscopy and assessment of findings
according to the list below:
1. Adrenal glands
2. All gross lesions
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Larynx (3 levels)
17. Lungs (5 lobes)
18. Lymph nodes (tracheobronchia l, mediastinal)
18. Lymph nodes (tracheobronchia l, mediastinal)
19. Lymph nodes (mesenteric)
20. Nasal cavity (4 levels)
21. Ovaries
22. Oviducts
23. Prostate gland
24. Peyer’s patches
25. Rectum
26. Sciatic nerve
27. Seminal vesicles
28. Spinal cord (cervical, thoracic, lumbar)
29. Spleen
30. Stomach (forestomach and glandular stomach)
31. Testes
32. Thymus
33. Thyroid glands
34. Trachea
35. Urinary bladder
36. Uterus
37. Vagina
Special attention was given on stages of spermatogenesis in the male gonads. Animals that died or were sacrificed in
a moribund state were processed histotechnically and assessed like control animals. Special stains of individual organs
of individual animals wer prepared if required.

Fetal examinations:

PUP STATUS AND LITTER SIZE AFTER BIRTH
Status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as
possible after birth. At the same time, the pups were also examined for gross-morphological changes.
PUP VIABILITY/MORTALITY
In general, a check was m ade for any dead or moribund pups twice daily on workdays or as a rule, only in the morning
on Saturdays, Sundays and public holidays. Pups, which died before the first determination of their status on the day
of birth, were defined as stillborn pups.
CLINICAL SIGNS
All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical
inspection of the dams. If pups showed particular findings, these were documented for each pup.
BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4. The body weight determined on PND 1 was also
used to determined runts. Those pups whose body weight was 25% below the mean body weight of the control group
(separately according to male and female pups) were defined as runts.
POSTMORTEM EXAMINATIONS OF PUPS
On PND 4, the pups were sacrificed under isoflurane anesthesia with CO2. After sacrificed pups were examined
externally and eviscerated, and their organs were assessed macroscopically. Pups that die or were sacrificed in a
moribund state were eviscerated and examined for possible defects and/or the cause of death. All pups without any
notable findings were discarded after their macroscopic evaluation.

Indices:

Reproductive indices
The testes, epididymides and ovaries of animals that died or had to be sacrificed intercurrently were fixed in 4%
buffered formaldehyde solution.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation
sites.

Details on maternal toxic effects:

Details on maternal toxic effects:
In males and females of test group 3 (6663 mg/m3 ), several clinical signs (unconsciousness, apathy, piloerection, reduced attention and unsteady gaits) were observed (during and after exposure) indicating narcotic effect of the test substance. In addition, animals showed some unspecific signs as alopecia and reduced fur care indicating bad general condition of the animals. Occasionally, gasping was observed in single animals. Moreover, reduced attention, apathy, reduced fur care, blood in bedding and vaginal discharge were observed in females of test group 3. Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls. The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3 ) and 3.

Dose descriptor:

NOAEC

Effect level:

2 561 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Effect level:

7 361 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEC

Effect level:

7 361 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

In males and females of test group 3 (6663 mg/m3 ), several clinical signs (unconsciousness, apathy, piloerection, reduced attention and unsteady gaits) were observed (during and after exposure) indicating narcotic effect of the test substance. In addition, animals showed some unspecific signs as alopecia and reduced fur care indicating bad general condition of the animals. Occasionally, gasping was observed in single animals. Moreover, reduced attention, apathy, reduced fur care, blood in bedding and vaginal discharge were observed in females of test group 3. Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls. The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3 ) and 3. The mean number of implantation sites, post implantation loss and liveborn pups was comparable between all test substance-treated groups and the controls. No effects were observed regarding developmental and teratogenicity.

Conclusions:

The Weight of Evidence approach was used to examine the reproductive toxicity of 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate.. NOAEC for maternal toxicity, developmental toxicity, and teratogenicity were determined to be 2561, 7361, and 7316 mg/m3, respectively, for t-amyl alcohol (CAS No. 75 -85 -4), one of the byproducts of hydrolysis of the registered test item, 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8).

Executive summary:

The registered substance, 00-tert-amyl-0-(2-ethylhexyl)monoperoxycarbonate (CAS No. 70833-40-8) is normally hydrolyzed into two major byproducts, 2-ethylhexanol (CAS No.104-76-7) and t-amyl alcohol (CAS No. 75-85-4). t-amyl alcohol may be further oxidized to diols and carboxylic acids and these reactions may be mediated by cytochrome p450-dependent oxidations. Therefore, available developmental toxicological data from t-amyl alcohol were used as the Weight of Evidence (WOE) approach to satisfy the developmental/teratogenic toxicity end point data requirements of 00 -tert-amyl-0 -(2 -ethylhexyl)monoperoxycarbonate.

 

A combined repeated dose and reproductive/developmenatal toxicity screening test (OECD 422) was reported to have been done under GLP conditions in male and female Wistar rats. The test article, t-amyl alcohol (CAS No. 75-85-4) was administered by inhalation (vapor) at nominal concentrations of 0, 732, 2561, and 7316 mg/m3 for test periods (premating, mating, and pregancy) in accordance with the OECD 422 guidelines.

In males and females of test group 3 (6663 mg/m3 ), several clinical signs (unconsciousness, apathy, piloerection, reduced attention and unsteady gaits) were observed (during and after exposure) indicating narcotic effect of the test substance. In addition, animals showed some unspecific signs as alopecia and reduced fur care indicating bad general condition of the animals. Occasionally, gasping was observed in single animals. Moreover, reduced attention, apathy, reduced fur care, blood in bedding and vaginal discharge were observed in females of test group 3. Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls. The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3 ) and 3. The mean number of implantation sites, post implantation loss and liveborn pups was comparable between all test substance-treated groups and the controls. The NAOEC for maternal toxicity was 2561 mg/m3. No effects were observed regarding developmental and teratogenicity at all doses..

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 20 September 2017 Experimental completion date: 22 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421 guideline for testing of chemicals adopted 29 July 2016: Reproduction/developmental toxicity screening test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate
Test item identity (including alternative names): tert-Amylperoxy-2-ethylhexylcarbonate
Trigonox 131
CAS number: 70833-40-8
Intended use: Industrial chemical
Appearance: Clear colorless liquid
Storage conditions: Frozen (-10 to -30C) in the dark
Supplier: Sponsor
Batch number: 1702442114
Retest date: 24 March 2019
Purity: 96.5%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS (UK) Limited.
Number of animals ordered 44 males and 48 females (92 in total; included 4 spare males and 8 spare females)
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization
Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment
Males: 84 to 90 days old.
Females: 98 to 104 days old.

Weight range of the animals at the start of treatment
Males: 319 to 358 g.
Females: 199 to 227 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.

Groups were adjusted to reduce inter-/intra-group variation.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Animal Care and Husbandry
Environmental Control

Rodent facility
Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing for mating; these were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) and discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Route of administration:

oral: gavage

Vehicle:

other: PEG300

Details on exposure:

Correction factor
Used as supplied; no correction factor applied.

Vehicle PEG300.

Method of preparation
O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was prepared for administration as a series of graded concentrations in the vehicle. Starting with the low concentration (50 mg/mL), the formulation was prepared by weighing out the required amount of test item, adding 40 to 50% of the final volume of vehicle and magnetically stirring until all the test item was uniformly mixed. The solution was made up to the required volume with the vehicle and stirred using a magnetic stirrer until homogenous.

The procedure was repeated for the mid and high dose (50 and 200 mg/mL).

Frequency of preparation
Weekly.

Storage of formulation
Refrigerated (2-8ºC).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis

Validated concentration range
20 mg/mL to 200 mg/mL
GLP Study: Harlan Laboratories Study Report: D45837 (homogeneity and stability establish for 4 hours at ambient temperature and 8 days refrigerated storage); 10 mg/mL validated as part of this study.
Stability assessment Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at a concentration of 10 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration during the first and last week of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in acetonitrile (50 mL).

Solutions for instrument calibration were prepared by appropriate dilution of the primarystandard using diluent and contained O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at nominal concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL and 250 μg/mL.

Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurate volume or weight where applicable) was dissolved in a suitable volume of acetonitrile. The extract was diluted using diluent to provide a solution containing O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at an expected concentration within the range 100 μg/mL to 200 μg/mL.

The concentration of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in the final solution was quantified by HPLC using UV detection.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (PEG300) with known amounts of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: XBridge Shield RP18, 75 × 4.6 mm, 3.5 μm
Column temperature: 45ºC
Sample temperature: 4ºC
Mobile Phase: ACN/water/ o-H3PO4 65/35/0.1 v/v/v
Flow rate: 1 mL/min
Needle wash: ACN/water 50/50 v/v
Detector wavelength: UV, 210 nm
Injection volume: 20 μL
Run time: 8 minutes
Approximate retention time: 4.3 minutes

Details on mating procedure:

Mating Procedure
Paired for mating After two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
3.6.6 Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Duration of treatment / exposure:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.

Frequency of treatment:

Daily

Duration of test:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males
10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Dose levels of 50, 250 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from an OECD 407 study conducted at 100, 300 and 1000 mg/kg/day (Harlan Laboratories Study Report D45837). In this study findings included:

• Reductions in food consumption at 1000 mg/kg/day
• Slightly high liver weights for females at 300 or 1000 mg/kg/day, with two animals exhibiting minimal centrilobular hepatocellular hypertrophy; considered to be metabolic adaption and therefore not adverse.
• Increased severity of hyaline droplets in the renal proximal tubules of males at 1000 mg/kg/day; this is considered to be an adverse finding in rats but has no toxicological significance in man.
• Forestomach mucosal necrosis for males at 100 mg/kg/day and for both sexes at 300 mg/kg/day or 1000 mg/kg/day; these were considered to be local injuries that resulted in subsequent adverse reactions.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for systemic toxicity was 1000 mg/kg/day. However the no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach.

On this study a high dose of 1000 mg/kg/day was anticipated to elicit parental effects in terms of food consumption, liver pathology and stomach lesions. A low dose of 50 mg/kg/day was selected with the aim to establish a NOEL, with an intermediate dose of 250 mg/kg/day to aid interpretation of any dose response.

Maternal examinations:

Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 females
Week 1 - daily
Week 2 - once

Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose
One to two hours after completion of dosing
As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

F0 females Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13

Body Weight
The weight of animals was recorded as follows:

F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before mating.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals
Weekly, before pairing.
For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Estrous Cycles
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.

For four days before scheduled termination (nominally Days 10-13 of lactation).

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination All surviving F0 adult males and females (no samples were obtained from animals which failed to litter or with litter death).

Terminal Investigations
Time of Necropsy

F0 females failing to produce a viable litter Day 25 after mating.
F0 females whose litter died before Day 13 On or after day the last offspring died.
F0 females Day 13 of lactation.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows for adult animals:
Tissue and regions examined

Abnormalities
Epididymides (caput, corpus and cauda)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulation gland)
Stomach
Thyroid
Uterus with cervix and oviducts

Ovaries and uterine content:

Duration of gestation
Time elapsing between the detection of mating and commencement of parturition.

Parturition observations
From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.
For apparently empty uterine horns The absence of uterine implantation sites was confirmed by
staining with ammonium sulphide [modification of the
Salewski staining technique (Salewski, E, 1964)].

Female whose litter died before Day 13 of lactation Mammary tissue appearance.

Fetal examinations:

Clinical observations
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size
Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all offspring.
Nipple/areolae count Day 13 of age - male offspring.


Thyroid Hormone Analysis
Blood samples were collected as follows:

Day 4 of age F1 offspring, two females per litter (where possible).
• one for T4 (serum)#
• one for TSH (plasma)
No females were allocated to these procedures if:
• The resultant live litter size fell below eight offspring.
• The resultant number of live females fell to less than three
If only four female offspring were available in a litter but the overall litter size was more than eight, one female pup was selected with priority given to the serum sample.
# priority was given to serum sample

Day 13 of age F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

Premature deaths
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 13 of age All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormal offspring were retained in appropriate fixative.
Thyroid glands were preserved from two offspring - one male and one female in each litter, where possible.

Statistics:

Please refer to "Any other information on materials and methods incl. tables"

Indices:

Mating Performance & Fertility

Group values were calculated for males and females separately for the following:

Percentage mating (%) = (No. of animals mating / Animals paired) x 100

Conception rate (%) = (No. of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (No. of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (No. of live litters born / Number pregnant) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to administration of the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight change for females during Week 1 of treatment was slightly low at 250 and 1000 mg/kg/day at approximately 57% of Controls; however this difference did not attain statistical significance. The overall weight gain for females for the two weeks before pairing, during gestation and lactation were unaffected by treatment at dose levels up to and including 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the first week of treatment females at 1000 mg/kg/day had slightly but not statistically significant low food consumption, approximately 89% of Controls. At 50 and 250 mg/kg/day food consumption was similar to Controls.
During gestation food consumption was considered unaffected by treatment.
During lactation the overall food consumption for females receiving 1000 mg/kg/day was marginally low when compared with Controls (approximately 93% of Controls), however the mean phase values were not statistically different from Controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Effects were only obersved in the stomachs of males

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related findings in the stomach of one female that received 250 mg/kg/day and one female that received 1000 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

Number of abortions:

not examined

Description (incidence and severity):

Not applicable, as rats do not abort.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The number of implantations and subsequently the live litter size on Days 1 and 4 of age (before selection of offspring for thyroid hormone sampling) were statistically significantly low at 1000 mg/kg/day when compared with concurrent Controls (implantations - p<0.01; litter size - p<0.05) and review of the historical control data showed that two litters on Day 4 were outside the 90 percentile range (7.7-14.4; n=74) . Litter size at 50 or 250 mg/kg/day was unaffected by parental treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Key result

Dose descriptor:

NOEL

Remarks:

Parental effects

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: acroscopic and microscopic changes in the stomach of parental animals at 250 and 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental effects

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: an overall parental NOAEL of 1000 mg/kg/day.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day body weight gain for male offspring from Day 4 to 7 of age was statistically significantly low when compared with Controls (p<0.05; approximately 83% of Controls). Female offspring at the same dose level showed marginally low weight gain over the same period (91 % of Controls), however this difference did not attain statistical significance. Offspring body weight gain from Day 7 to Day 13 of age and the overall weight gain from Day 1 to Day 13 of age was similar across the groups. Therefore this slight effect on offspring bodyweight gain at 1000 mg/kg/day was not considered to be adverse

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

• Low implantations and subsequent live litter size on Days 1 and 4 of age at 1000 mg/kg/day.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The number of implantations and subsequently the live litter size on Days 1 and 4 of age (before selection of offspring for thyroid hormone sampling) were statistically significantly low at 1000 mg/kg/day when compared with concurrent Controls (implantations - p<0.01; litter size - p<0.05). Litter size at 50 or 250 mg/kg/day was unaffected by parental treatment.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

. In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

At 1000 mg/kg/day one litter comprising of 13 offspring was found dead prior to Day 1 of age (litter no 140); the offspring weighed between 4.4 and 5.3 g. Macroscopic examination of the dam (no. 140) revealed 13 uterine implantation sites and pale/inactive mammary tissue. In the absence of any effect on offspring survival in the remaining litters this isolated litter death is considered to be unrelated to parental treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Offspring development

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

It was concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.

Executive summary:

 Summary

This study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with the administration of the test item [O‑(2‑ethylhexyl) O,O-tert-pentyl peroxycarbonate], an industrial chemical, by oral gavage, to Sprague Dawley rats for at least four weeks.

Three groups of ten male and ten female rats received test item at doses of 50, 250 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, PEG300, at the same volume dose as treated groups.

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation

length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results and Conclusion

Treatment ofO-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to parental animals at 50, 250 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance or fertility. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

Treatment related findings were restricted to:

·        Low implantations and subsequent live litter size on Days 1 and 4 of age at 1000 mg/kg/day. This effect was slight and in isolation with no effects on any other reproductive parameters or micropathology of the reproductive organs.

·        Macroscopic and microscopic changes in the stomach of parental animals at 250 and 1000 mg/kg/day. The microscopic findings of epithelial hyperplasia and submucosal infiltration of inflammatory cells in the non-glandular region of the stomach were considered to be indicative of a local irritant effect of the test item at 250 and 1000 mg/kg/day, with a dose-relationship in male animals; in males this correlated with gross findings of a thickened forestomach.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study of the registered substance.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 561 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study [OECD 422, inhalation) of t-amyl alcohol, abreakdown product of the registered substance

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Supportive evidence: The registered substance, O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate (CAS No. 70833-40-8) is normally hydrolyzed to two major byproducts, 2-ethylhexanol (CAS No.104-76-7) and t-amyl alcohol (CAS No. 75-85-4). Therefore, available reproductive/developmental toxicological data on t-amyl alcohol (OECD 422 inhalation study in rats) and 2 -ethylhexanol (OECD 414 dietary study in mice) were used as the Weight of Evidence (WOE) approach to support the developmental toxicity / teratogenicity end point requirements of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate.

Toxicity to reproduction: other studies

Additional information

As above.

Justification for classification or non-classification

The data are conclusive but not sufficient for classification.