Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A reproduction screening study is available to evaluate the reprotoxic potential of the registered substance (OECD 422).
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, and for females, during gestation and until day 4p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. Systemic toxicity was observed in parental males and females at the dose of 1000 mg/kg/d, however no adverse effects were observed on reproductive performance or on pups at the highest tested dose (1000 mg/kg/d).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 2015 -- 28 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 10 weeks old for the the males and 9 weeks old for the females on the first day of treatment
- Mean body weight at study initiation: for the males 398 g (range: 376 g to 417 g) and for the females 255 g (range: 236 g to 281 g).
- Housing: polycarbonate cages (Tecniplast 2154, 940 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 30 June 2015 to 28 August 2015

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Type of formulation
(visual observation): - solution in the vehicle.

Preparation procedure: - according to analysis homogeneity/stability study describing the preparation procedure for a range of concentrations covering the lowest and highest doses used in this study.

Frequency of preparation: - test item dose formulations
on the days of treatment

- vehicle dose formulations
based on the in-house procedures (frequency documented in study raw data).

Delivery conditions: - at room temperature.
- the test item dose formulations were administered within 5 hours after the end of their preparation.

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
Batch Nos.: MKBQ9948V and MKBS6944V
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occured or up to 3 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID.
Test item concentrations: remained within an acceptable range of variation compared to nominal values except for group 3 formulation in week 1 (but formulation given only once to the animals, formulation concentration was checked on week 2 and was correct).

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (5 weeks of treatment in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation (21-22 days),
- during lactation until Day 5 p.p. inclusive for lactating females,
- or until sacrifice for females with no evidence of mating or no delivery by Day 26 p.c..

Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Daily

Details on study schedule:

- No F1 parents (only 1 generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks

Dose / conc.:

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

Dose / conc.:

300 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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2 week toxicity study where groups of five male and five female Sprague-Dawley rats were given the test item in corn oil daily at 100, 300 or 1000 mg/kg/day.
During the second week of treatment, ptyalism was noted in both sexes at 300 mg/kg/day and at 1000 mg/kg/day and, dyspnea but mainly loud breathing were observed at 1000 mg/kg/day. Thin appearance, abdominal breathing and/or hunched posture were also noted during the second week of the study in one male at 300 and at 1000 mg/kg/day.
There were no adverse effects on body weight changes, food consumption or organ weights. There were no test item treatment-related macroscopic findings.

- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required)

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
Time schedule: once a day.

DETAILED CLINICAL SIGNS:
Once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT:
Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.

FOOD CONSUMPTION:
Time schedule: the quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p..

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.

Oestrous cyclicity (parental animals):

Fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below,
- special emphasis paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
Male animals: all surviving animals after the end of the mating period
Female animals: all surviving animals = Day 6 post-partum.
The following parent females were sacrificed by the same way without overnight fasting:
- females which did not deliver: on Day 26 p.c. (after a body weight recording to check for a possible un noticed delivery),
- female with no evidence of mating: 25 days after the end of the mating period as no delivery occurred.

ORGAN WEIGHTS: see table below


GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all parent animals

HISTOPATHOLOGY:
- on all tissues listed in the Tissue Procedure Table from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the control and high-dose groups (groups 1 and 4),
- forestomach from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the mid-dose group (group 3),
- on all macroscopic lesions of all groups,
- on all tissues listed in the tissue Procedure Table from the two animals (group 4) that died or were sacrificed prematurely,
- reproductive organs from the two pairs that did not conceive (group 3), to investigate possible causes.

Postmortem examinations (offspring):

SACRIFICE: on Day 5 post-partum

GROSS NECROPSY: all pups sacrificed on Day 5 p.p..

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

yes

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, ptyalism was observed during most of the treatment period, and loud breathing was also generally noted for a large part the treatment period. Loud breathing was considered to be adverse (serious clinical sign in 14/18 surviving animals for a long time generally) and ptyalism of minor toxicological importance.
One male and one female also experienced piloerection, round back, eyes half-closed, dyspnea and/or abdominal breathing for a few days at the end or at the beginning of the study, respectively.
One male was found to be emaciated in Week 2. This was also considered to be test item treatment-related but of minor toxicological relevance (single animal with this clinical sign).
At 300 mg/kg/day, ptyalism and loud breathing were recorded in 7 and 1 animals, respectively, and were considered to be related to the test item treatment and of minor toxicological importance in view of the absence of severity (ptyalism: not serious clinical sign) or of the limited incidence (loud breathing in 1/20 animals only).
There were no test item treatment-related clinical signs at 100 mg/kg/day.
The other clinical signs recorded in the study (chromorhinoirrhea, chromodacryorrhea, cutaneous lesion, area of hair loss) were not considered to be test item treatment-related: there were in limited incidences and are of common background of laboratory rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

At 1000 mg/kg/day, there were two deaths:
- one male prematurely sacrificed on Day 11 due to poor clinical condition [pallor of extremities (Day 11), cold to the touch (Day 11), loud breathing (from Day 2), abdominal breathing (from Day 10), dyspnea (from Day 10) and ptyalism (from Day 3) were recorded before death],
- one female found dead during pregnancy on Day 19 p.c.. Loud and abdominal breathing and ptyalism were observed for at least 12 days prior to its death.
Both deaths were considered to be related to the test item treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day:
- in males, when compared with controls, statistically significant lower mean body weight was noted from the first week of treatment. Mean body weight gain was indeed lower for the first 2 weeks of the study (mainly in the first week correlating with the decrease in mean food consumption). From the third week of treatment, mean body weight gain increased and the statistical difference in mean body weight disappeared. Mean body weight gain decreased again at the end of the treatment period,
- in females, when compared with controls, mean body weight gain had a tendency to be lower than in controls in the first week of treatment (correlating with the decrease in mean food consumption) but was higher than in controls in the second week of treatment. Mean body weight was not affected throughout the study.
These variations from controls were considered to be test item treatment-related but non-adverse (because of the low amplitude of body weight changes).

At 100 and 300 mg/kg/day, there were no test item treatment-related effects on mean body weights and mean body weight changes. The statistically significant higher mean body weight gain in males at both doses from Days 15 to 29 were considered to be not relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean food consumption was toxicologically significantly lower during the first week of treatment in both sexes. Mean food consumption had also a tendency to be lower than in controls during gestation but this was considered to be of no toxicological significance in the absence of statistical significance and because of the low amplitude of variation.

There were no effects on mean food consumption at 100 and 300 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

yes, section details in section 7.5.1

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

yes, section details in section 7.5.1

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

yes, section details in section 7.5.1

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):

yes, section details in section 7.5.1
No changes on sexual organs were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

yes, section details in section 7.5.1

- Genital tracts of animals which did not conceive
One male had unilateral marked atrophy/degeneration of testicular tubular epithelium which may have contributed to the absence of conception. This lesion was considered to be spontaneous, in view of the presence of similar finding in controls.
One male and two females had no noteworthy findings in the reproductive tract.

The minimal cortical hypertrophy seen in one male treated at 1000 mg/kg/day was considered to be secondary to the stress.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

In the control group, there was one female (often blocked in diestrus) that did not mate with its two males, and at 300 mg/kg/day, there were 2/10 females that were not pregnant although mated.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on mating, fertility and gestation indexes and on the mean pre-coïtal time.
There were no test item treatment-related effects on mean duration of gestation, mean pre- and post-implantation losses and mean number of pups delivered.
At 1000 mg/kg/day, the slightly lower mean number of corpora lutea, implantations, number of pups delivered and the slightly higher mean pre-implantation loss compared with controls were particularly due to one female.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity (systemic and local)

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: two deaths and the loud breathing present in most animals at 1000 mg/kg/day,

Dose descriptor:

NOEL

Remarks:

reproductive performance

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no effects on pup mortality and viability.
At 300 mg/kg/day, viability index was slightly lower than in controls due to one female with 1 dead and 2 cannibalized pups on Day 1 p.p..

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on mean pup body weight and body weight gain at the beginning of the lactation period.
At 300 mg/kg/day, mean pup body weight at birth was lower in both sexes than in controls. Then the pups had a tendency to gain more weight than the other groups and pup mean body weight on Day 5 p.p. was thus comparable to the other groups. The difference in pup mean body weight at birth was not considered to be test item-related in absence of dose-relationship.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test-item treatment related findings.
At 100 mg/kg/day, one litter had 3 pups with no milk in the stomach at scheduled sacrifice, and one pup with dilated ureter.
In prematurely dead pups, autolysis and absence of milk in the stomach observed at necropsy were of common background in prematurely dead rat pups in this kind of study.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on pup sex ratio in test item-treated groups as it is close to 50% what is normally expected.
The control group had a low pup sex ratio.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

toxic effects on progeny

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Based on the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the two deaths and the loud breathing present in most animals at 1000 mg/kg/day,
- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects on mating, fertility and delivery data up to 1000 mg/kg/day,
- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects in pups up to 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 5 post-partum (p.p.).

This study provided information on:

- male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 p.p..The test item was administered at dose-levels of 100, 300 and 1000 mg/kg/day in the vehicle (corn oil). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked in study Weeks 1, 3 and 6 and one more time (in Week 2) for groups 1 and 3.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recordedonce a week during premating, mating (food consumption not during mating) and gestation (0, 7, 14 and 20 p.c.) periods and during lactation on Days 1 and 5 p.p..

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p.The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p..

The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p..Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on several organs from five males and five females in the control and high-dose groups and from the dead animals, on forestomach from five males and five females in the mid-dose group, on reproductive organs from pairs that did not conceive although mated, and on all macroscopic lesions.

The pups were sacrificed on Day 5 p.p. and a macroscopic post-mortem examination was performed. No tissue was preserved.

Results

 

Chemical analyses

There was no test item found in control formulation samples and the test item concentrations in the administered dose formulations analyzed were within an acceptable range of variationsexcept in Week 1 for group 3 dose formulation which was found at +12.6%. However, the formulation was given only once to the animals and thus did not have an impact on the study conclusion.

 

Mortality

At 1000 mg/kg/day, there were two deaths considered to be test item treatment-related: one male prematurely sacrificed on Day 11 for poor clinical conditions and one female found dead on Day 19 p.c.. Both rats had at least loud breathing for several days before death. At pathology,there were no clear causes of death/moribundity. The dead animals had hyperplasia of forestomach with hyperkeratosis (grossly described as white discoloration in the female) probably related to the test item administration, and findings in the thymus and adrenals (female) which were stress-related.

There were no premature deaths at 100 and 300 mg/kg/day.

 

Clinical signs

At 1000 mg/kg/day, loud breathing and ptyalism were observed for a long time during the treatment period. Loud breathing was considered as adverse (serious clinical sign recorded in 14/18 surviving animals generally for a long time). Two animals also experienced piloerection, round back, eyes half-closed, dyspnea and/or abdominal breathing for a few days, and one other male was emaciated in Week 2.

At 300 mg/kg/day, ptyalism (7/20 animals) and loud breathing (1/20 animal) were considered to be of minor toxicological significance.

There were no test item-related clinical signs at 100 mg/kg/day.

Body weight

At 1000 mg/kg/day,when compared with controls, mean body weight was lower in males for the first half of the treatment period (down to -7%, p<0.01) along with a low mean body weight gain (mainly during the first treatment week: +9 g vs.+40 g in controls, p<0.001). From the third week of treatment, mean body weight gain increased (+53 g vs +30, p<0.001) but was nul in the last week of treatment (vs.+19 g in controls). In females, at the same dose, mean body weight was not affected; there was a tendency towards a low mean body weight gain in the first week of treatment (+5 g vs. +13 g), followed by a higher mean body weight gain the week after (+20 g vs. +11 g, p<0.05). These variations of low magnitude were considered to be non-adverse.

There were no test item-related effects at 100 and 300 mg/kg/day.

Food consumption

At 1000 mg/kg/day, when compared with controls, mean food consumption was lower during the first week of treatment in both sexes (-21/-22%, p<0.001/0.01) correlating with the concurrent decreased in mean body weight gain.

There were no test item-related effects at 100 and 300 mg/kg/day.

Pathology

At terminal sacrifice, there were no test item-related mean organ weight differences and no test item-related gross findings.

At microscopy, slight non-adverse test item-related erosion/ulcer, with squamous cell hyperplasia and hyperkeratosis, were noted in 1/5 females treated at 1000 mg/kg/day.

Mating, fertility and delivery data

There were no test item treatment-related effects on mating, fertility and gestation indexes and on the mean pre-coïtal time, as well ason mean duration of gestation, mean pre- and post-implantation losses and mean number of pups delivered.

Pup data

There were no test item treatment-related effects in pup mortality, clinical signs, mean body weight, sex ratio and at necropsy at the beginning of the lactation period.

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 5 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

Based on the experimental conditions and results of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the two deaths and the loud breathing present in most animals at 1000 mg/kg/day,

- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects on mating, fertility and delivery data up to 1000 mg/kg/day,

- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects in pups up to 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This screening test is reliable, performing according to the OECD guideline no.422 (klimisch score of 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined 28 -day repeated toxicity and reproduction screening test - OECD 422 (Bentz 2016)

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 5 post-partum (p.p.). This study provided information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of theconceptusand parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5p.p..The test item was administered at dose-levels of 100, 300 and 1000 mg/kg/day in the vehicle (corn oil). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

At 1000 mg/kg/day, there were two deaths considered to be test item treatment-related: one male prematurely sacrificed on Day 11 for poor clinical conditions and one female found dead on Day 19 p.c.. Both rats had at least loud breathing for several days before death. At pathology,there were no clear causes of death/moribundity. The dead animals had hyperplasia of forestomach with hyperkeratosis (grossly described as white discoloration in the female) probably related to the test item administration, and findings in the thymus and adrenals (female) which were stress-related.

At 1000 mg/kg/day, loud breathing and ptyalism were observed for a long time during the treatment period. Loud breathing was considered as adverse (serious clinical sign recorded in 14/18 surviving animals generally for a long time). Two animals also experienced piloerection, round back, eyes half-closed, dyspnea and/or abdominal breathing for a few days, and one other male was emaciated in Week 2. At 300 mg/kg/day, ptyalism (7/20 animals) and loud breathing (1/20 animal) were considered to be of minor toxicological significance.

At 1000 mg/kg/day,when compared with controls, mean body weight was lower in males for the first half of the treatment period (down to -7%, p<0.01) along with a low mean body weight gain (mainly during the first treatment week: +9 gvs.+40 g in controls, p<0.001).From the third week of treatment, mean body weight gain increased (+53 gvs+30, p<0.001) but was nul in the last week of treatment (vs.+19 g in controls). In females, at the same dose, mean body weight was not affected; there was a tendency towards a low mean body weight gain in the first week of treatment (+5 gvs.+13 g), followed by a higher mean body weight gain the week after (+20 gvs.+11 g, p<0.05). These variations of low magnitude were considered to be non-adverse.

At 1000 mg/kg/day, when compared with controls, mean food consumption was lower during the first week of treatment in both sexes (-21/-22%, p<0.001/0.01) correlating with the concurrent decreased in mean body weight gain.

At terminal sacrifice, there were no test item-related mean organ weight differences and no test item-related gross findings.

There were no test item treatment-related effects on mating, fertility and gestation indexes and on the mean pre-coïtal time, as well ason mean duration of gestation, mean pre- and post-implantation losses and mean number of pups delivered. There were notest item treatment-relatedeffects in pup mortality, clinical signs, mean body weight, sex ratio and at necropsyat the beginning of the lactation period.

Based on the experimental conditions and results of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the two deaths and the loud breathing present in most animals at 1000 mg/kg/day,

- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects on mating, fertility and delivery data up to 1000 mg/kg/day,

- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day, based on the absence of test item treatment-related effects in pups up to 1000 mg/kg/day

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

-

Justification for classification or non-classification

Based on the results of the screening test on reproduction, no classification for the registered substance is required for reprotoxicity according to the Regulation EC no.1272/2008.