tert-butyl hydroperoxide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-2-methylpropane

EC Number:

211-309-7

EC Name:

2-ethoxy-2-methylpropane

Cas Number:

637-92-3

Molecular formula:

C6H14O

IUPAC Name:

Tert-Butyl Ethyl Ether

Details on test material:

ETBE, >98% pure, batch no. S02-08-159-I3 (S02-08-159-I3/1, S02-08-159-I3/2), colourless liquid, supplier: TOTAL France S.A., Paris-la-Défense, France (purified by SEPAREX, Champigneulles, France)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Standard as per guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: time-mated female Sprague Dawley rats (Charles River Laboratories, L'Arbresle, France)
Age at start of treatment: approx. 7 wk (mean bw 203 g males, 157 g females)
Acclimation period: 6 d
Housing: individually housed, stainless steel cages (wire mesh bottom) for duration of study
Diet: A04 C pelleted maintenance diet (UAR, Villemoisson, France), ad libitum
Water: deionised water, ad libitum
Assignment to treatment groups: computer-generated weight randomisation

The animal room conditions are set as follows:
- temperature: 22 ± 2°C
- relative humidity: 50 ± 20%
- light/dark cycle: 12h/12h (7:00 - 19:00)
- ventilation: about 12 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ETBE was administered to groups of 25 male and 25 female rats by gavage at doses of 0 (corn oil; 4 ml/kg bw), 250, 500 or 1000 mg/kg/day for 7 days/week as follows:

F0 generation:
- males: from 10 wk before mating, during 2 wk mating period and until sacrifice (after weaning of F1 pups), total duration of treatment: 18 weeks.
- females: from 10 wk before mating, during 2 wk mating period, pregnancy and lactation until day 21 post-partum, total duration of treatment: 18 weeks.

F1 and F2 generations:
- as above, commencing day 22 post-partum

Details on mating procedure:

Mating followed a 10 wk treatment period. Dosing of F0 animals began at age 7 wk, with mating from age approx. 17 wk. Dosing of F1 animals began at weaning when the animals were 22 d old, with mating from age approx. 13 wk.

Females were monitored for estrous stage (microscopic examination of fresh vaginal lavage, stained with methylene blue) each morning during the last 3 weeks of the pre-mating period and during the mating period (until mating confirmed by presence of a vaginal plug or sperm in a vaginal lavage).

Females were paired overnight with males from the same dose group until mating had occurred or 14 days had elapsed, whichever occurred first. Parental F1 animals were selected from different litters to avoid brother-sister matings.

Pregnant females were housed individually, allowed to litter normally and raise progeny until weaning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions were prepared weekly (based upon stability data demonstrating no losses of ETBE over 9 d) and samples taken for analysis (GC) to confirm received dose. Stability and homogeneity studies returned satisfactory results (all analysed concentration within 10% of nominal).

Duration of treatment / exposure:

F0 generation:
- males: from 10 wk before mating, during 2 wk mating period and until sacrifice (after weaning of F1 pups)
- females: from 10 wk before mating, during 2 wk mating period, pregnancy and lactation until day 21 post-partum

F1 and F2 generations:
- as above, commencing day 22 post-partum

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were selected in agreement with the Sponsor on the basis of a previously conducted study (CIT/Study No. 24168 RSR). The results demonstrated a slight but statistically significant decrease in body weight for pregnant female rats given 1000 mg/kg/day, and ptyalism (excess salivation) over the course of the study in both sexes given 1000 mg/kg/day. No effect on mating, pregnancy, lactation or litter data parameters was noted at 50, 250, 500 and 1000 mg/kg/day in this preliminary investigation. Consequently, dose-levels of 250, 500 and 1000 mg/kg/day were selected for use in the main two-generation study. It is noted that 1000 mg/kg/day is considered a limit dose for this type of investigation (OECD Guideline 416).

Positive control:

Not a guideline requirement

Examinations

Parental animals: Observations and examinations:

Parental animals were observed at least twice daily for clinical signs, morbidity or mortality. Body weight and food consumption was recorded weekly for males, and at least weekly for females, commencing during the pre-mating period.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined by microscopic examination of a fresh vaginal lavage (stained with methylene blue), each morning as follows:
- during the last 3 weeks of the pre-mating period;
- during the mating period, until mating was confirmed.

Sperm parameters (parental animals):

Sperm collected (under isoflurane anaesthesia, prior to terminal sacrifice) from F0 and F1 parental males for quantitative evaluation of:
- epididymal sperm motility (x40 magnification), sperm count (cauda sperm count, Malassez cell) and sperm morphology (eosin staining, x40 magnification)
- homogenisation resistant testicular sperm (Neubauer cell)

Litter observations:

Total litter size, number of live/dead pups and sex of each pup was recorded as soon as possible after birth, and the pups observed daily for clinical signs. Anogential distance was measured (both generations) on day 1. Any gross malformations were noted. The size of each litter was adjusted to 4 males and 4 females on post-partum day 4. Pup body weight was recorded on days 1, 4, 7, 14 and 21 post-partum.

Reflex development was recorded in F1 and F2 pups as follows:
- surface righting reflex on day 5 post-partum
- cliff avoidance on day 11 post-partum
- air-righting reflex on day 17 post-partum

Acoustic startle response and pupil constriction reflex were assessed in F1 pups at 4 wk of age. Spontaneous motor activity was evaluated in F1 pups twice, at weekly intervals, when the animals were 7-8 wk old.

Males monitored between 32-47 d old for preputial separation, females monitored between 28-40 d old for vaginal opening (pups from both generations).

Postmortem examinations (parental animals):

Parental animals sacrificed (carbon dioxide) after weaning of the litters (that is, between day 22-25 post-partum), weighed, and subject to a gross internal examination (including examination of the uterus for implantation scars).

Following organs weighed:
Males:
- testes (separately), epididymides (separately), prostate, seminal vesicles together with coagulating glands, brain, liver, kidneys, spleen, pituitary gland, thyroids with parathyroids, adrenals
Females:
- uterus, ovaries, brain, liver, kidneys, spleen, pituitary gland, thyroids with parathyroids, adrenals,

PRESERVATION OF TISSUES:

The following organs preserved from all F0 and F1 parental animals:
ovaries and oviduct,
uterus (with cervix and horns)
vagina
testis (right)
epididymis (right)
seminal vesicles
prostate
coagulating glands
pituitary gland
adrenal gland
A vaginal smear taken from all females and stained (Harris Schorr staining).
Any abnormal tissue found at necropsy in the weanling pups was preserved.

EXAMINATION OF TISSUES

The following tissues from control and high dose F0 and F1 animals stained with haematoxylin-eosin prior to microscopic
examination:
any macroscopic abnormalities
ovaries and oviducts (including qualitative assessment of
primordial follicle population)
uterus (with cervix and horns)
vagina
epididymis
seminal vesicles
prostate
coagulating glands
pituitary gland
adrenal gland
The vaginal smear taken from females at terminal sacrifice also examined microscopically.

The testis from all treated males plus the controls was stained with haematoxylin/PAS and subject to a detailed histopathological examination (including an assessment of retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells).
Any macroscopic abnormalities present in the pups born to F0 and F1 mothers were prepared for histopathological evaluation.

Postmortem examinations (offspring):

Three pups/sex/litter born to the F0 and F1 mothers subject to gross necropsy at weaning. Brain, spleen and thymus removed and weighed.

Statistics:

Mean values were compared by one-way ANOVA and Dunett's test (mean values being considered as normally distributed and variances being considered homogenous). Percentage values were compared by the Fisher exact probability test.
Mean percentages of motility and morphology sperm parameters were Arcsine transformed and then compared by ANOVA. Mean values for anogenital distance, ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of the individual pup's body weight were compared by analysis of variance.

Reproductive indices:

F0 generation:

Mating indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).
Fertility indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).

F1 GENERATION

Mating indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).
Fertility indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).

Offspring viability indices:

F0 litters/F1 pups:
0 250 500 1000 mg/kg/d
Viability index on LD4 (%) 97.6 92.9 82.3 97.7

F1 litters/F2 pups:
0 250 500 1000 mg/kg/d
Viability index on LD4 (%) 97.6 94.8 97.0 92.9

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

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

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

PARENTAL/ADULT OBSERVATIONS:
F0 PARENTAL GENERATION
No deaths in either sex during the pre-mating, mating or gestation periods. One female given 250 mg/kg bw/d and three females given 500 mg/kg bw/d sacrificed during the first week of lactation because their entire litters died or were in poor condition. Considered unrelated to treatment by studydirector since no comparable findings in the high dose group.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to unpalatable nature of the dosing solutions by study director.
Body weight gain decreased significantly in mid (-29%; P<0.01) and high (-22%; P<0.001) males on study days 85-113 (i.e. final 28 days of treatment, following mating). Considered treatment-related by study director. Female body weights unremarkable. Food intake not adversely affected by treatment in either sex.

F1 PARENTAL GENERATION
One control male sacrificed due to poor condition on study day 36, one female given 1000 mg/kg bw/d found dead on day 47, and one low dose female and one high dose female sacrificed early because their entire litters were found dead a few days after birth. These findings considered unrelated to treatment by the study director.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to the unpalatable nature of the dosing solutions by study director.
Body weight and food consumption unaffected by treatment in both sexes.

REPRODUCTIVE DATA FOR PARENTAL GENERATIONS:
F0 PARENTAL GENERATION
The majority of pregnancy and parturition parameters unremarkable, with implantation, fecundity, neo-natal loss, gestation and delivery unaffected by treatment. The exception was a slightly greater post-implantation loss in the low and mid dose groups, reflecting litters found dead or sacrificed prematurely due to poor clinical condition. Considered unrelated to treatment by study director since no similar findings in high dose group.

F1 PARENTAL GENERATION
Pregnancy and parturition parameters unremarkable, with implantation, fecundity, post-implantation loss, neo-natal loss, gestation and delivery unaffected by treatment. A single pregnant female from the 250 mg/kg bw/d group did not deliver, and was found to have only a single implantation site at necropsy. Considered unrelated to treatment by study director since only one animal from the low dose group affected.

SPERM ANALYSES FOR F0 AND F1 PARENTAL MALES
No statistically significant or dose-related changes in seminology data for F0 and F1 males. A slightly lower testicular sperm head count noted in all treated F0 males appeared to be the consequence of a high control value. Overall, sperm parameters unaffected by treatment in both generations at all dose-levels.
------ mg/kg bw/d ------
0 250 500 1000
F0 MALES
Epididymal sperm
Spermatozoa (10^3/mm^3) 923 938 938 918
Motility (%) 99.7 100 98.6 97.6
Normal morphology (%) 93 93 97 96

Testicular sperm
Sperm heads (10^6/g testis) 148 109 108 110
Sperm production (10^6/g/d) 18.8 17.9 17.1 18.0

F1 MALES
Epididymal sperm
Spermatozoa (10^3/mm^3) 725 673 701 688
Motility (%) 84.6 87.1 93.3 88.3
Normal morphology (%) 84 86 86 88

Testicular sperm
Sperm heads (10^6/g testis) 100.6 97.8 105.3 99.8
Sperm production (10^6/g/d) 16.5 16.0 17.3 16.4

NECROPSY AND HISTOPATHOLOGY
F0 GENERATION PARENTS
Absolute and relative liver weights significantly increased in high dose males, and correlated with liver enlargement noted during necropsy and hepatocellular hypertrophy (considered adaptive response to treatment by the study director) in livers from three animals selected for histopathological examination. Liver weights from females given 1000 mg/kg/day slightly (increased 4-6%) but non-significantly increased. Kidney weights significantly increased in high dose males and correlated with the presence of microscopic acidophilic globules in cortical tubular epithelium from 5 of 6 animals selected for microscopic examination (considered related to alpha2u-globulin accumulation by study director). Kidney weights also significantly increased in male rats given 500 mg/kg/day (no microscopic examination performed).

Summary table: percentage change in F0 liver and kidney weights
--- Males --- -- Females --
250 500 1000 250 500 1000
Liver
- absolute +2 +2 +17** -1 +4 +6
- relative +3 +6 +24** +10 +8 +4

Kidney
- absolute +11 +15** +21** -1 +2 +5
- relative +11 +18** +28** +9 +5 +3

All other organ weight and macroscopic findings consistent with those observed in untreated rats of this strain and age, and considered of no toxicological importance by the study director.

F1 GENERATION PARENTS
Absolute and relative liver weights significantly greater in males given 500 or 1000 mg/kg/day and correlated with liver enlargement recorded at necropsy. Microscopic examination of liver tissue from two selected high dose males revealed slight or moderate hepatocellular hypertrophy, considered an adaptive response to treatment by the study director. Absolute and relative kidney weights significantly increased in males given 1000 or 500 mg/kg/day, with slight to marked accumulation of acidophilic globules (considered related to alpha2u-globulin accumulation by study director) detected microscopically in tissue from one selected mid dose male and four selected high dose males.

Summary table: percentage change in F1 liver and kidney weights
--- Males --- -- Females --
250 500 1000 250 500 1000
Liver
- absolute +0 +14** +27** +1 +3 +10**
- relative +0 +11** +25** +3 +6 +9*

Kidney
- absolute +10 +22** +58** +4 +3 +11**
- relative +10** +19** +58** +6 +6 +10**

All other organ weight and macroscopic findings consistent with those observed in untreated rats of this strain and age, and considered of no toxicological importance by the study director.

MICROSCOPIC EXAMINATION OF REPRODUCTIVE ORGANS
F0 GENERATION
Minimal degeneration of seminiferous tubules present in two control and two high dose animals, with desquamated spermatocytes present in control (8/25 animals) and treated high dose animals (1/25 animals). These findings commonly recorded in rats of this strain and age and considered of no
toxicological importance by the study director. Interstitial mononuclear cell aggregation and subacute prostatitis of the prostate and spermatic granuloma in the epididymides occurred with equal incidence and severity in both control and treated animals in this study, and considered of no toxicological importance by the study director.
Microscopic examination of the ovaries, uterus and vagina fromcontrol and high dose females revealed morphological changes consistent with a regular estrous cycle in both control and treated animals. Quantitative analysis of the primordial and growing follicles in the ovaries showed no perceptible differences between control and treated animals.

F1 GENERATION
A reduced number of tailed and round spermatids, spermatocytes, and spermatogonia present in 3/25 low dose males, 1/24 mid dose males and 3/25 high males versus 0/25 in the controls. In the majority of instances, changes correlated with seminiferous tubules lined with Sertoli cells only (minimal severity), with oligospermia or aspermia in the epididymides of the males receiving 1000 mg/kg/day (epididymides not examined in the low and intermediate dose-level groups). Minimal focal degeneration of germinal epithelium observed in 1/24, 1/25 or 1/25 males from the control, low- or high-dose groups, respectively. Minimally or slightly degenerated/necrotic cells observed in the lumen of the testis from 9/24 control males, 4/25 low dose males, 8/24 mid dose males and in 6/25 high dose males. Although some of these observations not present in the controls, the study director considered there was no evidence of a dose-response relationship in their incidence and/or severity, and that similar findings can occur spontaneously in the untreated rats of this strain and age. Consequently, findings considered unrelated to treatment.
Microscopic examination of the ovaries, uterus and vagina from control and high dose females revealed morphological changes consistent with a regular estrous cycle in both control and treated animals. Quantitative analysis of the primordial and growing follicles in the ovaries showed no perceptible differences between control and treated animals.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

adult toxicity

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Remarks on result:

other: Generation not specified (migrated information)

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: treatment had no effect on mating, fertility, gestation, fecundity, delivery or sperm parameters. No effects were observed on the progeny from delivery until weaning

Remarks on result:

other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Parental/adult observations:
F2 GENERATION:
One control female found dead on day 25, necropsy signs consistent with mis-dosing.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to the unpalatable nature of the dosing solutions by study director.
Body weight and food consumption unaffected by treatment in both sexes.

LITTER SIZE AND PUP PARAMETERS DURING LACTATION
F0 LITTERS/F1 PUPS:
The number of litters delivered by the F0 dams and number of F1 pups per litter unaffected by treatment. At 250 mg/kg/d and 500 mg/kg/d there was a slight increase in the number of pups that died during the first 4 days of lactation, however findings for the high dose group were unremarkable and the findings were considered unrelated to treatment by the study director. Viability and lactation indices were unaffected by treatment at all dose levels.
0 250 500 1000 mg/kg/d
Litters obtained 23 21 22 25
Pups/dam 14.3 14.1 14.9 14.2
Viability index on LD4 (%) 97.6 92.9 82.3 97.7
Total decedent pups (LD1-21) 18 35 64 10
Lactation index (%) 94.6 91.7 96.1 99.0

F1 LITTERS/F2 PUPS
The number of litters delivered by the F1 dams and number of F2 pups delivered per litter unaffected by treatment. At 250 mg/kg/d and 1000 mg/kg/d, the number of pups that died during the lactation period was slightly higher than controls during the first 4 days of lactation. Considered unrelated to treatment by study director since only one (particularly large) litter per dose group affected. Viability and lactationindices unaffected by treatment at all dose levels.
0 250 500 1000 mg/kg/d
Litters obtained 21 21 22 20
Pups/dam 13.7 13.7 13.7 14.0
Viability index on LD4 (%) 97.6 94.8 97.0 92.9
Total decedent pups (LD1-21) 11 17 9 21
Lactation index (%) 97.6 98.8 100.0 99.3

PUP CLINICAL SIGNS, BODY WEIGHTS AND EXTERNAL FINDINGS
F1 PUPS
Pups from one low dose litter and three mid dose litters emaciated in appearance, cold to the touch or dehydrated, and were found dead or prematurely sacrificed for ethical reasons. Similar findings also observed in four surviving pups from three litters at 250 mg/kg/day, three surviving pups from one litter at 500 mg/kg/day and six pups from four different litters at 1000 mg/kg/day. Most of these pups displayed lower body weight. Since incidence was not dose related and did not correlate with lower mean group body weight gain, findings considered unrelated to treatment by the study director.
Body weight and body weight gain unaffected by treatment.
No gross external abnormalities present in F1 pups born to F0 mothers.

F2 PUPS
Tremors and ataxia present from day 15 post-partum in one pup from the 250 mg/kg/day group (sacrificed for humane reasons). Other clinical signs (necrosis of the tail or of the limbs,cold to the touch, emaciated appearance) were low in incidence, not dose-related and randomly distributed between the litters and treatment groups.
No effect on body weight or body weight gain noted at 250 and 500 mg/kg/day, while high dose pups exhibited a transient non-significant decrease (-11%) in body weight gain during the first 4 days after birth.
At 1000 mg/kg/day, absence of tail (acaudia) observed at birth in two female pups from two different litters, with anal atresia also present in one of these pups. The incidence of acaudia (2/280 pups; 0.7%) was outside the historic control range for the laboratory (minimum = 0.0%; maximum = 0.05%) data from 16 studies, October 2000-December 2003) but close to the background incidence recorded in the MARTA [*] historical control database (0.31% for 22147 fetuses from 1575 control litters during the period 1990-2004). The study director concluded that no clear relationship to treatment was established. No historical control data were available from the laboratory performing the study for anal atresia, however the observed incidence (1/280 pups; 0.35%) was close to that recorded in the MARTA historical control database (0.32% for 22147 fetuses from 1575 control litters during the period 1990-2004). Occurrence of anal atresia in this investigation considered spontaneous in origin by the study director.

[* A historical control database of preclinical developmental teratology and reproductive toxicity parameters, a joint project of MARTA and MTA; www.hcd.org]

Comment: No instance of acaudia or anal atresia in off-spring from dams treated with ETBE at a dose of 1000 mg/kg/day during a preliminary dose-range finding study (zero incidence in 164 fetuses from 12 litters and 144 pups from 11 litters; CIT study 24168 RSR, October 2003; summarised in this IUCLID dataset) or a developmental toxicity study (zero incidence in 258 fetuses from 22 litters; CIT study 24860 RSR, January 2004; summarised in this IUCLID dataset).

PUP ANOGENITAL DISTANCE, REFLEX DEVELOPMENT AND SEXUAL MILESTONES:
F1 PUPS:
Anogenital distance, the ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of pup body weight not significantly altered by treatment.
Mean AGD values (in mm, SD in brackets) were:
0 250 500 1000
Males 4.71 (0.43) 4.62 (0.33) 4.47 (0.39) 4.64 (0.30)
Females 2.84 (0.36) 2.77 (0.24) 2.63 (0.26) 2.68 (0.37)

No evidence of any adverse treatment-related changes in surface righting, cliff avoidance and air righting (95 to 100% positive responses). (Observations performed on F1 pups only.)
Mean age at preputial separation unaffected by treatment (i.e. occurred on day 35, 34, 35 or 35 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Mean age for vaginal opening unaffected by treatment (i.e. occurred on day 34, 34, 35 or 33 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Auditory function (acoustic startle reflex) and visual function (pupil constriction reflex) at 4 weeks of age, and spontaneous locomotor activity (measured twice at 7 and 8 weeks old) unaffected by treatment. (Observations performed on F1 pups only)

F2 PUPS:
Anogenital distance, the ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of pup body weight not significantly altered by treatment. Mean AGD values (in mm, SD in brackets) were:
0 250 500 1000
Males 4.57 (0.39) 4.56 (0.42) 4.54 (0.42) 4.63 (0.38)
Females 2.80 (0.35) 2.72 (0.29) 2.82 (0.29) 2.90 (0.33)

No evidence of any treatment-related effect on surface righting, cliff avoidance and air righting (between 96.0 and 99.4% positive responses).
Mean age at preputial separation similar in the control and the treated groups (i.e. occurred on day 36, 35, 35 or 36 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Mean age for vaginal opening similar in the control and the treated groups (i.e. occurred on day 35, 34, 34 or 33 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively). Although pups from two high dose dams displayed acaudia and anal atrasia, anogenital distance was normal.

NECROPSY AND HISTOPATHOLOGY:
F1 PUPS:
No treatment-related or statistically significant changes in pups found dead or sacrificed early, or in pups subject to scheduled necropsy at weaning.

F2 pups:
No treatment-related or statistically significant changes present in pups found dead or sacrificed early, or in pups subject to scheduled necropsy at weaning.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

In the two-generation study ptyalism was observed at all dose levels (i.e., from 250 mg/kg bw/day onwards). The study authors considered this effect related to unpalatable nature of the dosing solutions / to the unpalatable 'taste' of ETBE. In the EU RAR this effect is not taken into account as an adverse effect, therefore, this effect a not considered a significant health effect.

Applicant's summary and conclusion

Conclusions:

The following No Observed Adverse Effect Levels were established from the study:
Systemic toxicity in the adult (parental) F0 and F1 generations: NOAEL = 250 mg/kg body weight/day (based on body weight and organ weight changes at higher treatment levels).

Ptyalism (excess salivation) was noted in all treated animals (LOEL = 250 mg/kg body weight/day but was not considered to represent an adverse effect of treatment.

Fertility, gonadal function, reproductive performance, parturition and lactation in the parental generations, and development of the off-spring to weaning or sexual maturity: NOAEL = 1000 mg/kg body weight/day (the highest dose tested).

Executive summary:

The test item, ETHYL TERTIARY BUTYL ETHER (ETBE), CAS No. 637-92-3, was
administered daily by oral gavage to male and female Sprague-Dawley rats at 250, 500 and 1000 mg/kg/day, commencing 10 weeks prior to mating and continuing through mating and gestation until the end of lactation in both the F0 and F1 generations. Progeny of the F1 generation (F2 pups) were treated from weaning until sexual maturity.

For all generations, ptyalism (excessive salivation) was observed with a dose-related trend in both males and females. At 1000 mg/kg/day, F0 males showed significantly lower body weight gain at the end of the dosing period. Liver weight was significantly increased in males only, with slight to moderate centrilobular hepatocellular hypertrophy in tissue from animals subject to microscopic
examination. Kidney weights were also significantly increased in F0 parental males, with acidophilic globules detected after microscopic examination. There were no adverse findings for F0 pups. Significantly greater food consumption during the lactation period was the only finding of note in F0 parental females.

Liver and kidney weights were significantly increased in F1 parental males.
Body weight gain of pups born to mothers from the F1 generation was slightly but
non-significantly lower than the controls on post-partum days 1-4 (no comparable finding in F0 litters). Two pups born to mothers from the F1 generation exhibited gross external malformations (absence of tail with anal atresia also present in one pup), however the incidence of these findings was comparable to laboratory or external historical control data. Neither malformation was present in 566 pups or fetuses from 45 litters from dams treated with ETBE at 1000 mg/kg body weight/day as part of a dose-range finding study and a developmental toxicity
study performed at this laboratory. It was concluded that the findings from the present study were therefore most probably unrelated to treatment with the test item.

No effects were noted in the F2 generation at 1000 mg/kg/day. At 500 mg/kg/day, significantly lower body weight gain was noted at the end of the dosing period in F0 parental males together with significantly increased kidney weights. Liver and
kidney weights were statistically significantly increased in F1 parental males, whereas body weight was unaffected. No effects were noted in the F2 generation.
At 250 mg/kg/day, no relevant findings were observed in the F0, F1 and F2 generations.

Based on these observations, the following No Observed Adverse Effect Levels were established from the study:
Systemic toxicity in the adult (parental) F0 and F1 generations: NOAEL = 250 mg/kg body weight/day (based on body weight and organ weight changes at higher treatment levels).

Ptyalism (excess salivation) was noted in all treated animals (LOEL = 250 mg/kg body weight/day but was not considered to represent an adverse effect of treatment.

Fertility, gonadal function, reproductive performance, parturition and lactation in the parental generations, and development of the off-spring to weaning or sexual maturity: NOAEL = 1000 mg/kg body weight/day (the highest dose tested).