tert-butyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Read-across, OECD TG 416, GLP compliant, K1, rats (oral: gavage), NOAEL fertility, systemic toxicity = 400 mg/kg bw/d) conducted on a structural analoge

 

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 16, 2008 - December 02, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on October 15, 2010; GLP certified by Landesamt für Umwelt. Wasserwrtschaft und Gewerbeaufsicht (October 2009)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number of test material: 012231eda0
- Purity: 99.9 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator; avoiding temperatures >35°C
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was proven by reanalysis. The stability in the vehicle at room temperature over a period of 7 days was investigated analytically before the beginning of the study. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. The homogeneous distribution of test substance in the vehicle was demonstrated.
- Expiry date: 16 Jan 2009

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

This rat strain was selected since extensive historical control data were available for Wistar rats and the rat is the preferred animal species for reproduction studies according to test guidelines.

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
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males/females: not specified
- Fasting period before study: not specified
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)

Analytical verification of doses or concentrations:

yes

Remarks:

Range of analytical values (see table 1 in "Any other information on materials and methods incl. tables").

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Duration of treatment / exposure:

until one day before sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

450 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 generation parental animals: 25
F1 generation parental aniumals: 25

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).

OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 and F1 males of all dose groups.
The following parameters were determined:
• sperm motility
• sperm morphology
• sperm head count (cauda epididymis)
• sperm head count (testis)
Sperm motility examinations and the preparation of the specimens for sperm morphology were carried out in a randomized sequence.
Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
- Standardization of litters was not performed in litters with ≤ 8 pups

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

other:
sexual maturation: All female F1 pups selected to become the F1 parental generation females (25/group) were evaluated daily for vaginal patency beginning on PND 27. At the day of vaginal opening, the body weight of the respective animals was determined. All male F1 pups selected to become the F1 parental generation males (25/group) were evaluated daily for preputial separation beginning on PND 38. At the day of preputial separation, the body weight of the respective animals was determined.

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS (please see also table 2 in "Any other information on materials and methods incl. tables")
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.

Statistics:

see table 3 in "Any other information on materials and methods incl. tables"

Reproductive indices:

- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations for males and females:
(except gestation and lactation period)All male and 7/25 female F0 parental animals of the high-dose group (400 mg/kg body weight/day) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). This symptom was initially observed during study week 5.
The temporary salivation is considered to be test substance-induced. From the the
temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding

Clinical observations for females during gestation of F1 litters:
9/25 high-dose (400 mg/kg bw/d) F0 females showed transient salivation during major parts of the gestation period (GD 0 - 16 and GD 20).
There were no clinical findings in F0 females during the gestation period for the F1 litter at low- and mid-dose levels (50 and 150 mg/kg bw/d). Three sperm positive females of test group 00 (Nos. 104, 110 and 117 - 0 mg/kg bw/d), test
group 01 (Nos. 128, 141 and 146 - 50 mg/kg bw/d), test group 02 (Nos. 166, 173 and 175 – 150 mg/kg bw/d) and one female of test group 03 (No. 193 - 400 mg/kg bw/d) did not deliver F1 pups.

Clinical observations for females and offspring during lactation of F1 litters:
7/24 high-dose (400 mg/kg bw/d) F0 females showed transient salivation during major parts of the lactation period (PND 0 - 8, PND 10, PND 14 - 16 and PND 18 - 21). The F0 females of low- and mid-dose groups (50 and 150 mg/kg bw/d) did not show any clinical findings during the lactation period of the F1 pups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

males and females (except gestation and lactation):
During study week 11, one high dose F0 male (No. 98 - 400 mg/kg body weight/day) was found dead, after showing hypothermia, salivation and labored respiration. Pathological examination revealed pleuritis and pericaditis as the cause of death, most likely secondary to a gavage error.
During lactation period, two high dose F0 females (Nos. 185 [PND 18] and 197 [PND 13]) were found dead. There were no macroscopic or histolopathologic findings that could explain the death of these two animals

females during gestation of F1 litters:
9/25 high-dose (400 mg/kg bw/d) F0 females showed transient salivation during major parts of the gestation period (GD 0 - 16 and GD 20).

females and offspring during lactation of F1 litters:
7/24 high-dose (400 mg/kg bw/d) F0 females showed transient salivation during major parts of the lactation period (PND 0 - 8, PND 10, PND 14 - 16 and PND 18 - 21).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly increased or decreased body weight change in the low-dose females during study weeks 0 - 1 and weeks 4 - 5, in the mid-dose females during study weeks 1 - 2 and weeks 4 - 6 and in the high-dose females during weeks 4 - 6 was considered as spontaneous in nature.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose F0 males (400 mg/kg bw/d) was statistically significantly decreased (up to 7%) during premating weeks 5 - 10.
Food consumption of the low- and mid-dose F0 males (50 and 150 mg/kg bw/d) was generally comparable to the respective controls throughout the entire study.
Food consumption of the high-dose F0 females (400 mg/kg bw/d) was statistically significantly decreased (6 - 10%) during several segments of premating (weeks 1 - 3, 5 - 8, and 9 - 10), gestation (gestation days 0 - 7) and lactation (postnatal days 4 - 7). The mid-dose F0 females (150 mg/kg bw/d) had a slightly but statistically significantly decreased food consumption during premating weeks 1 - 2 (about 5%) and gestation days 0 -7 (about 7%). Food consumption was comparable to the control animals during the remaining premating, gestation and the entire lactation period.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings were either single observations, or were similar in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

The male fertility index ranged between 88% and 96% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The female fertility index varied between 88% (test groups 00 - 02) and 96% (test group 03). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The gestation index was 100% in all test groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (13.4 / 12.1 / 13.2 and 12.4 implants/dam in test groups 0 - 3 (0, 50, 150 and 400 mg/kg bw/d)). Furthermore, there were no indications for test substance induced intrauterine embryo-/fetolethality since the post-implantation loss did not show any statistically significant differences between the groups. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% (test group 00, 02 and 03) and 100% (test group 01). Moreover, the number of stillborn pups was comparable between the groups.

The mean number of delivered F1 pups per dam and the rate of liveborn and stillborn pups were not affected by the test substance.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: not accompanied by clinical, clinical pathological and pathological signs

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicity up to and including the highest tested dose

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity up to and including the highest tested dose

Critical effects observed:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females (except gestation and lactation):
24/25 male and 12/25 female F1 parental animals of the high-dose group (400 mg/kg body weight/day) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). This symptom was initially observed during study week 6 in males and towards the end of the study in females. The temporary salivation is considered to be test substance-induced. From the the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F1 generation parental animals at low- and mid-dose levels (50 and 150 mg/kg bw/d). Other clinical findings like labored respiration, microphthalmia, poor general state, palpable mass in the neck and throat region in some low- and mid-dose individuals were considered as spontaneous in nature and not as test substance-related.

Clinical observations for females during gestation of F2 litters:
5/25 high-dose (400 mg/kg bw/d) F1 females showed transient salivation during
major parts of the gestation period (GD 4, 6 - 7, 9, and 11 - 12). There were no clinical findings in F1 females during the gestation period for the F2 litter at low- and mid-dose levels (50 and 150 mg/kg bw/d). The clinical finding micropthalmia (one high dose female) was considered as spontaneous in nature and not test substance-related. One sperm positive female each of test group 10 (No. 302), test group 11 (No. 347 - 50 mg/kg bw/d) and test group 13 (No. 377 - 400 mg/kg bw/d) did not deliver F2 pups.

Clinical observations for females and offspring during lactation of F2 litters:
There were no clinical findings in F1 females during the lactation period for the F2 litter at all dose levels (50, 150 and 400 mg/kg bw/d).
The microphthalmia (one high dose female) and palpable mass in the throat region (one low dose female ) were considered as spontaneous in nature and not test substance-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose parental males had statistically significantly lower body weights during weeks 0 - 5 (up to 17%) and 10 - 11 (up to 6%). Mean body weight gain of these parental males was comparable to the control group throughout the entire study. Mean body weights and mean body weight gain of the F1 parental males in the low- and mid-dose groups (50 and 150 mg/kg bw/d) were comparable to the concurrent control throughout the entire study. High dose parental females had statistically significantly lower body weights during premating weeks 0 - 1 (up to 16%). High dose body weights were comparable to the control animals during the remaining premating, and the entire gestation and lactation periods. Mean body weight gain of these parental females was comparable to the control group throughout the entire study. Mean body weights and mean body weight gain of the F1 parental females of the low and mid-dose groups (50 and 150 mg/kg bw/d) was comparable to the control animals during premating, gestation and lactation.
The statistically significantly increased body weight gain in the high dose females (400 mg/kg bw/d) during premating weeks 1 - 2 and premating weeks 0 - 10 was considered as spontaneous in nature.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose F1 males (400 mg/kg bw/d) was statistically significantly decreased (about 14%) during premating weeks 0 – 1 and 8 - 10 (up to 7%). Food consumption of the low- and mid-dose F1 males (50 and 150 mg/kg bw/d) was generally comparable to the respective controls throughout the entire study. Food consumption of the high-dose F1 females (400 mg/kg bw/d) was statistically significantly decreased (7-10%) during several segments of premating (weeks 0-1 and 9-10), and gestation (gestation days 0–14). High-dose food consumption was comparable to the control animals during the remaining gestation and the entire lactation period. Food consumption of the F1 parental females of the low and mid-dose groups (50 and 150 mg/kg bw/d) was comparable to the control animals during premating, gestation and lactation

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

mean absolute weights of the following organs were significantly changed:
liver, kidneys, brain, cauda epididymis
The increase of liver weights of the 150 and 400 mg/kg dose group in females and the increase in kidney weights in all treated females were regarded to be related to treatment. The increase of the cauda epididymidis weight in all treated males was considered incidental and not treatment-related as there was no histopathologic correlate observed. The decrease of brain weights in the 400 mg/kg dose group male animals was regarded to be a consequence to the reduced terminal body weight.

mean relative weights of the following organs were significantly changed:
liver, kidneys, cauda epididymis
The increase of relative liver and kidney weights of female animals of all treatment groups were regarded to be related to treatment. The increase in liver weight in males of the 400 mg/kg dose group and of kidney weights in males of the 150 and 400 mg/kg dose group were also regarded to be treatment related. The increase in epididymides weights in all treated males were thought to be reflective to the lower body weight. In addition, there was no histopathological correlate corresponding to the weight decrease in the 400 mg/kg dose group.

There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions observed in test animals occurred singularly. They were considered to be spontaneous lesions in origin and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant deviations between controls and animals of the 400 mg/kg dose group.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

For all F1 parental males, which were placed with females to generate F2 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F1 parental males within the scheduled mating interval for F2 litter.
One control male (No. 224), one low dose male (No. 229) and one high-dose male (No. 299) did not generate F2 pups. Thus, the male fertility index ranged between 96% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility. The apparently infertile male rats (Nos. 224, 229, and 299) did not show histopathological findings that could explain infertility.

The female mating index calculated after the mating period for F2 litter was 100% in all test groups. The fertility index varied between 96% (test groups 10, 11 and 13) and 100% (test group 12). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility and do not show any relation to dosing. The mean duration of gestation values varied between 22.0 and 22.2 days without any relation to dosing. The gestation index was 100% in all dose groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.2 / 12.5 / 11.7 and 11.9 implants/dam in test groups 10 -13 (0, 50, 150 and 400 mg/kg bw/d)). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (see Tab. IA-062), and the mean number of F2 pups delivered per dam remained unaffected (11.1 / 12.0 / 11.0 and 10.9 pups/dam at 0, 50, 150 and 400 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% in all test groups. Moreover, the number of stillborn pups was comparable between the groups.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: not accompanied by clinical, clinical pathological and pathological signs

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicity up to and including the highest tested dose

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity up to and including the highest tested dose

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100% (test group 01) and 99% (test group 00, 02 and 03). The lactation index indicating pup mortality on PND 4 - 21 varied between 100% (test group 01), 99% (test group 00), 97% (test group 02) and 92%** (p≤0.01, test group 03). The statistically significantly decreased lactation index in the high-dose group (400 mg/kg bw/d) was caused by the non-test-substance related death of two dams on PND 13 and 18, the litters of which had to be sacrificed. These litter losses were considered as spontaneous in nature. Thus, the test substance did not influence pre-weaning F1-pup survival in any of the treated groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A temporary decrease of mean body weight gain was noted for the F1 high-dose pups (400 mg/kg bw/d) on PND 4 – 7. Considering the absence of significant differences in the weights themselves these decreases were regarded as incidental and not treatment-related.

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:

effects observed, treatment-related

Description (incidence and severity):

Vaginal opening:
Each female F1 pup, which was selected to become a parental F1 female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 28, the last was PND 42. The mean age at vaginal patency was 31.6, 33.0, 33.2* (p≤0.05), and 33.9** (p≤0.01) days in the control and 50, 150 and 400 mg/kg body weight/d test groups.
All mean age values in the low and mid-dose groups were within the historical control range the slightly, higher age values in these groups were mainly driven by one outlier each, which became sexually mature only on PND 42. The mean age value in the high-dose group was just above the upper historical limit. This
minimal, apparent increase in the high-dose group is considered to be secondary to the lower female body weight during major phase of sexual maturation. The majority of the control females (17/25) and high-dose females (15/25)
became sexually mature when they weighed between 80 and 100 grams. The control body weight curve crosses the 80 grams line at about 26 days of age and the 100 grams line at about 34 days of age. For the high-dose body weight curve this is the case at about 29 and 36 days of age, respectively. This 2-3 days difference in weight gain corresponds almost exactly to the difference in the age when vaginal patency occurred.

Preputial separation:
Each male F1 pup, which was selected to become a F1 parental male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 39, the last was PND 49. The mean number of days to reach the criterion in the control and 50, 150 and 400 mg/kg body weight/day test groups was 41.6, 41.4, 41.6, and 42.5 days, indicating that male sexual maturation was not influenced by the test substance.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant effects on the brain weights:
97% of control for males and females in the dose group 400 mg/kg bw/d,
95% and 96% for males in the dose groups 150 and 400 mg/kg bw/d
These marginal differences in absolute brain weights reflect the normal biological variation in this strain of rats as they are comparable to the historical control data. Relative brain weights remained unchanged. The findings are neither adverse nor toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as situs inversus, incisors sloped, supernumerary lung lobe, empty stomach, small kidney, enlarged kidney, dilated renal pelvis and small testis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Therefore, these findings were not considered to be associated to the test substance. The number of affected pups per litter was statistically significantly increased in test group 02 (150 mg/kg bw/d). However, the incidence of 3.1% is well within the historical control range and there is no dose-response relationship. Thus, no association to the treatment was assumed.

Other effects:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The mean number of delivered F2 pups per dam and the rate of liveborn and stillborn pups were not affected by the test substance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic toxicity up to and including the highest tested dose

Critical effects observed:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Female pup No. 14 of control dam No. 312 had a multiple malformed left hindlimb and for female pup No. 8 of high-dose dam No. 398 a lateral position was recorded on one occasion during lactation (PND 19).

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 98% (test group 11 - 50 mg/kg bw/) and 99% (test group 10, 12, 13 - 0, 150, 400 mg/kg bw/d). The lactation index indicating pup mortality on PND 4 - 21 varied between 98% (test group 11 and 12), 99% (test group 13) and 100% (test group 10).
Thus, the test substance did not influence pre-weaning pup survival in any of the treated groups (50, 150 and 400 mg/kg bw/d). The statistically significantly increased number of cannibalized pups in test group 11 was considered as spontaneous in nature.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Temporary decreases of mean body weight change were noted in the F2 male and/or female pups in test group 13 (400 mg/kg bw/d) on PND 1 - 4 and PND 4 – 7 as well as in test group 11 (50 mg/kg bw/d) on PND 14 - 21. Considering the absence of significant differences in the weights themselves these decreases were regarded as incidental and not treatment-related

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Single F2 pups showed some spontaneous findings at gross necropsy such as incisors sloped, hemorrhagic thymus, diaphragmatic hernia, empty stomach, dilated renal pelvis, dilated ureter, small testis, haematoma and multiple malformed hindlimb. Every pup necropsy finding occurred without relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The sex distribution and sex ratios of live F2 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic toxicity up to and including the highest tested dose

Critical effects observed:

not specified

Reproductive effects observed:

not specified

- Analytics:

The various analyses:

• demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
• confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
• showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group	Nominal Dose (mg/kg bw/d)	Analytical Dose (mg/kg bw/d) [minimum]	Analytical Dose (mg/kg bw/d) [maximum]	% Nominal Dose [minimum]	% Nominal Dose [maximum]
00 / 10	0	0	0
01 / 11	50	43.40	46.61	86.8	93.2
02 / 12	150	132.90	169.80	88.6	113.2
03 / 13	400	359.20	379.90	89.8	95.0

 

Conclusions:

The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested. The NOAEL for systemic toxicity, in the F1 and F2 progeny of the test substance is 400 mg/kg bw/day, the highest dose tested.

Executive summary:

The study was performed according to OECD TG 416 in compliance with GLP (reliability 1). The test item was administered to groups of 25 male and 25 female healthy young Wistar rats (P, parental generation) as an aqueous preparation by stomach tube at dosages of 0, 50, 150, and 400 mg/kg bw/d. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0, 50, 150, and 400 mg/kg bw/d post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals. Control parental animals were dosed daily with the vehicle (1% carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

 

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P0 generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversely effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation was not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of the test substance via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any general toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Under the conditions of the present reliable 2-generation reproduction toxicity study the NOAEL(no observed adverse effect level) for general, systemic toxicity was 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOAEL for fertility and reproductive performance for the F1 parental rats was 400 mg/kg bw/d, the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data availability:

Sufficient data regarding the reproductive toxicity of the test item can be obtained by read-across from a reliable 2-generation reproductive toxicity study on a structural analogue as well as a reliable reproductive/developmental screening study perfoemd with the target substance. No further testing is proposed.

 

In an OECD 416 study (GLP, reliability 1; 2010) the structural analogue has recently been tested in  in rats, in which both, parental and F1 animals were dosed by gavage with 0; 50; 150 and 400 mg/kg bw/d.

In summary, in mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance. The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL 50 mg/kg bw/day for the P parental rats and NOEL 150 mg/kg bw/d for F1 parental rats).

In detail: The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0, 50, 150 and 400 mg/kg bw/d. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0, 50,150 and 400 mg/kg bw/d post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals. Control parental animals were dosed daily with the vehicle (1% carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid). The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group. High-dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain. High-dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversely effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed. Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathological lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathological findings. It is not regarded to be an adverse effect. There were no indications from clinical examinations as well as gross and histopathology, that the administration of the test item adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behaviour, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility. All data recorded during gestation and lactation in terms of embryo-/foetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day). The NOEL for general, systemic toxicity was determined to be 50 mg/kg bw/day for the P parental rats, based on adverse effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females. The NOEL for general, systemic toxicity was determined to be 150 mg/kg bw/day for the F1 parental rats based on adverse effects on food consumption and body weight at the highest tested dose. However, these effcts were not regarded as any true systemic toxicity because no accompanying clinical, clinical pathological and pathological signs were noted in previous and the present studies. The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested.

 

Supporting study: In an reliable OECD 421 study (reliability 2, 2008) the test substance was administered at 0 (control group: aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100, 300 and 1000 mg/kg to Sprague- Dawley SPF rats, for 14 days before mating and throughout the mating period until the day before autopsy in males (42 days), and for 14 days before mating and throughout the mating period and gestation period until day 4 of nursing in females (42-48 days), and the repeated dose toxicity and reproductive and developmental toxicity were investigated. In the parent animals, test substance administration had no observable effect on estrous cycle, days required until copulation, copulation index, insemination index or conception index. Furthermore, test substance administration had no observable effect on delivery index, gestation period, number of corpora lutea, number of implantation marks, implantation index, stillbirth index, number of liveborn pups, livebirth index or sex ratio, and no abnormalities in nursing condition were observed. Therefore, test substance administration was thought to have had no effect on reproductive functions such as copulation ability in males or females, or on insemination ability or conception ability, or on pregnancy maintenance, delivery and nursing behavior, etc. of dams, even in the 1000 mg/kg group. In the liveborn pups, no changes due to test substance administration were observed in body weight at birth or on day 4 of nursing, in males or females, and no changes due to test substance administration were observed on external examination at birth, or in the autopsy findings on day 4 of nursing, or in the viability index. It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance in males and females was less than 100 mg/kg/day, and that the no-observed effect dose with respect to reproduction and development toxicity in male and female parents and pups was 1000 mg/kg/day.

 

In a supportive OECD Guideline 422 screening study (reliability 1, 1998), the structural analogue in sesame oil was given by oral gavage to 10 male and 10 female rats at doses of 0, 30, 100, 300, or 1000 mg/kg/day (Ito et al., 1998). Male rats were dosed for 44 days and female rats were dosed from 14 days prior to mating through Day 3 of lactation. Treatment-related decreases in body weight and food consumption were observed in high dose (1000 mg/kg/day) animals only. Relative to reproductive performance, there were no treatment-related effects on any endpoint of mating or fertility at any dose level. There were no treatment-related effects on gestation index, gestation length or number of pups per litter. There were no treatment-related deaths during delivery, nor treatment-related effects on offspring viability or the ratio of male to female pups. There were no adverse effects on the reproductive organs at any dose of male (testes, epididymides, prostate, seminal vesicles) and female (ovaries, uterus, cervix, vagina) rats shown by gross and histopathological examination. Observed decreases in numbers of corpora lutea and implantations in parental females in the high dose group were the only effects ascribed to treatment.

Thus, based on effects observed in parental females in the 1000 mg/kg/day dose group, the NOAEL for reproductive toxicity is considered to be 1000 mg/kg/day for parental males and 300 mg/kg/day for parental females.

Effects on developmental toxicity

Description of key information

Read-across, OECD 414, GLP, K1, rabbit, oral: NOAEL maternal toxicity = 100 mg/kg bw/d, NOAEL maternal developmental toxicity = 1000 mg/kg bw/d, NOAEL developmental toxicity fetuses = 300 mg/kg bw/d

 

Read-across, similar to OECD 414, GLP not specified, K2, rat, inhalation: NOAEC maternal toxicity = 100 ppm, NOAEC maternal developmental toxicity = 1200 ppm, NOAEC developmental toxicity fetuses = 300 ppm

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 May 2008 - 24 Jun 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) Rheinlandpfalz; date of inspection: 8.06.2005 and 25.-27.07.2005

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Batch number of test material: 1271192067
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, temperatures >30°C were avoided
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Analytical verifications of the stability of the test substance in 1% CMC for a period of at least 4 hours at room temperature were carried out before the study was initiated.
- Solubility and stability of the test material in the solvent/vehicle: The homogeneous distribution of test substance in the vehicle (1% CMC) was demonstrated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Concentration of the aqueous preparations: 1000, 3000 and 10000 mg/100 ml

FORM AS APPLIED IN THE TEST
- aqueous test substance preparations

OTHER SPECIFICS
- Physical state: liquid/colorless, clear

Species:

rabbit

Strain:

Himalayan

Remarks:

Crl:CHBB[HM]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 18-23 weeks
- Weight at study initiation: 1901-2589 g
- Fasting period before study: no
- Housing: single housing
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)

IN-LIFE DATES: From: June 2008 To: July 2008

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at daily intervals, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The graduated flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.

Details on mating procedure:

The animals were paired by the breeder (time-mated animals), the day of pairing was designated as gestation day (GD) 0. Presumed pregnant animals were supplied one day after mating; this day was referred to as GD 1 and the following day as GD 2.

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

orally (by gavage) in an ascending dose once a day at approximately the same time of day (in the morning)

Duration of test:

until GD 29

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 time-mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.
- Dose selection rationale: The doses were chosen at the request of the sponsor.
- Rationale for animal assignment (if not random): After the supply of the animals on GD 1, they were assigned to the different test groups according to a randomization plan (Nijenhuis and Wilf, 1978) and on the basis of their body weights.

Maternal examinations:

MORTALITY: Yes
Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 1-29).

CLINICAL EXAMINATIONS: Yes
A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 1-29).
- Food consumption: The food consumption was determined daily on GD 2–29.
- Body weight data: All animals were weighed on GD 1, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

BODY WEIGHT: Yes
All animals were weighed on GD 1, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.

FOOD CONSUMPTION: Yes
The food consumption was determined daily on GD 2–29.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus and placenta

OTHER:
Corrected (net) body weight gain : The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

Ovaries and uterine content:

On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined.
After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ.
After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.

Statistics:

- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Indices:

The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100;

The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula:
(number of corpora lutea – number of implantations)/(number of corpora lutea) x 100;

The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula:
(number of implantations – number of live fetuses)/(number of implantations) x 100

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-associated clinical findings such as reduced/no defecation and abortions were observed in the high-dose group. Although such findings are not uncommon in rabbits, they were, in these particular cases, most likely a consequence of the massively reduced food consumption in these rabbits. Taking this fact and the high incidence of findings in the high-dose group into account they are thus considered to be related to the treatment. By contrast the low frequency of findings like reduced defecation or blood in bedding does not suggest a relationship to the treatment in the low- and mid-dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Seven high-dose females had to be sacrificed after abortion on several days towards the end of the treatment period (GD 24-28). Before abortion, the food consumption in all affected individuals was distinctly reduced and they showed adverse clinical symptoms like reduced and no defecation. These 7 premature sacrifices because of abortions are considered to be a consequence of test substance-induced maternal toxicity. There were no test substance-related or spontaneous mortalities in any other group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the high-dose rabbits (1000 mg/kg bw/d) were statistically significantly decreased on GD 21, 23, 25 and 29. At termination (GD 29) the body weight of the surviving pregnant high-dose rabbits was about 5% below the concurrent control. The average body weight gain of these rabbits was also statistically significantly reduced at a number of treatment intervals (GD 6-9, 16-19, 19-21, 25-28). The mean body weight gain of the high-dose rabbits was significantly reduced by about 47% during the treatment period. The mean body weights and the mean body weight gain of the low- and mid-dose rabbits (100 and 300 mg/kg bw/d) were not significantly different from the concurrent controls throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the high-dose females (1000 mg/kg bw/d) was distinctly and statistically significantly reduced during the almost entire treatment period (GD 6-27 and 28-29). During the treatment period (GD 6-28) the total average food consumption of the highdose
rabbits was about 44% below controls. Additionally, a particularly massive decrease of individual food consumption was noted for all high-dose does which aborted. During the days prior to abortion the food consumption was almost disrupted in these animals. The females of the mid-dose group (300 mg/kg bw/d) also had a statistically significant lower food consumption in a number of intervals during the treatment (GD 8-9, 13-16, 17-18, 19-20, 21-22, and 26-29). During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 18% below controls. The food consumption of the low-dose does (100 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 300 and 1000 mg/kg bw/d levels is considered to be related to the treatment.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean gravid uterus weight in the high-dose group (1000 mg/kg bw/d) was statistically significantly reduced (about 21% below the control).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, 7/25 high-dose does had stomach erosions, 5/25 high-dose does had no feces in the small intestine and 3/25 high-dose does had watery feces in the intestines. These findings are related to the markedly reduced food consumption and are considered to be treatment-related. In the mid- and low-dose groups, only incidental, not test substance-related findings were noted in single females of each of these test groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Corrected (net) body weight gain: Mean carcass weights were comparable among all groups. The corrected (net) body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was statistically significant (about 54 %) below the concurrent control at the high-dose (1000 mg/kg bw/d) level. Although not statistically significant the net body weight gain was still 37% below control at the mid-dose (300 mg/kg bw/d) level. These reductions in net body weight gain arer considered to be related to the treatment. No adverse effect on net body weight gain was noted in the low-dose group.
- Uterus weight: The mean gravid uterus weight in the high-dose group (1000 mg/kg bw/d) was statistically significantly reduced (about 21% below the control). The mean gravid uterus weights of test groups 1 and 2 (100 or 300 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

7 abortions were observed in the high-dose group (1000 mg/kg bw/d)

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The resorption rate (and hence the postimplantation loss) was statistically significant higher in the high-dose group (1000 mg/kg bw/d). This apparent effect was mainly caused by two does of this dose group which had only resorptions but no viable fetuses in the uterus and contributed to the average with 100% resorption rates.
- for details please refer to table 1 in section" Any other information on results incl. tables"

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

The litter losses in these two does are considered to be a direct consequence of maternal toxicity. In particular the markedly reduced food consumption after mid-gestation affected these animals in the same manner as the animals with abortions. All other does of the high-dose group did not show any changes of gestational parameters which were outside the normal range for this strain.
- for details please refer to table 1 in section" Any other information on results incl. tables"

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

A significant increase of late resorptions could be observed in the highest dose group.
- for details please refer to table 1 in section" Any other information on results incl. tables"

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 100% in all test groups including controls (0, 100, 300 or 1000 mg/kg bw/d).

Other effects:

no effects observed

Details on maternal toxic effects:

The litter losses in these two does are considered to be a direct consequence of maternal toxicity. In particular the markedly reduced food consumption after mid-gestation affected these animals in the same manner as the animals with abortions. All other does of the high-dose group did not show any changes of gestational parameters which were outside the normal range for this strain. There were no test substance-related and/or biologically relevant differences between the low- and mid-dose groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

number of abortions

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weight of the high-dose group (1000 mg/kg bw/d) was statistically significant below the corresponding control value (-10%). The mean fetal weights of the low- and mid-dose groups (100 or 300 mg/kg bw/d) were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between these groups and the controls

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations, some associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). With the exception of cleft palate all external malformations were present in the historical control data. Each of the findings affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data. External variation: One external variation (paw hyperflexion) occurred in a few fetuses of the mid- and high-dose
groups and the control. An association of this finding to the treatment is not assumed. External unclassified observations: No unclassified external observation was recorded in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal malformations: Malformations of the fetal skeletons were noted in all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). All individual skeletal malformations were present in the historical control data. With the exception of severely fused sternebrae (bony plate), which was observed at a slightly higher incidence, the frequencies of those malformations were comparable to the historical control data. With regard to the total malformation incidences, in particular the litter incidence, neither statistically significant differences between treated groups and control nor a dose-response relationship were observed. Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher exclusively in the
high dose group on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent and outside the historical control in the high dose group (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain, but considering the overall higher rate of skeletal variations in the top-dose group they may be correlated to the treatment. Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 100; 300 and 1000 mg/kg bw/d). All individual soft tissue malformations were present in the historical control data at comparable frequencies. Neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed. Fetal soft tissue variations: A number of soft tissue variations such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted. Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, discolored kidney, hemorrhagic ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1 and 2 (0; 100 and 300 mg/kg bw/d). A relation to dosing is not present. Therefore, a test substance induced effect is not assumed.

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were comparable to the controls.;

One external variation (paw hyperflexion) occurred in a few fetuses of the mid- and high-dose groups and the control. The incidences as listed in Tab. 4.3.2.2.1. did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.;

Fetal external unclassified observations: No unclassified external observation was recorded in this study.

Details on embryotoxic / teratogenic effects:

- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. The most salient of these malformations affected the head/skull (malformed skull bones, cleft palate), the heart/great vessels (various defects), gall bladder (absent), kidneys (absent, small) and the sternebrae (fused to a bone plate). All malformations are present in the historical control data, with the exception of cleft palate. No dose-response relationship is evident for the individual malformations. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) the overall incidences are comparable to the concurrent and historical controls, whereas the overall incidence is slightly above the controls at the high-dose level. Importantly, as a number of morphological structures of different ontogenic origin is affected these various fetal findings do not form a distinct and consistent malformation pattern. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) all fetal and litter incidences for these variations and the corresponding rates of affected fetuses/litter were comparable to the control whereas at the high-dose level a slightly higher rate of affected fetuses per litter was noted. However, all incidences including the high-dose incidence can be found at a comparable frequency in the historical control data. A spontaneous origin is assumed for soft tissue and unclassified skeletal cartilage observations, which were observed in several fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 and 1000 mg/kg bw/d) and the control. Distribution and type of these findings did not suggest relation to treatment.
- for details please refer to tables 2-4 in section" Any other information on results incl. tables"

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Postimplantation loss and resorption data

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d	HCD (range)
Postimplantation loss	Mean %	8.9	8.2	7.1	23.1*	12.1 (4.6 – 50.0)
Total resorptions	Mean	0.6	0.6	0.5	1.6*	0.7 (0.4 – 3.1)
	Mean %	8.9	8.2	7.1	23.1*	12.0 (4.6 – 50.0)
Late resorptions	Mean	0.2	0.3	0.3	0.9*	---
	Mean %	2.0	4.5	3.9	14.2*	---

HCD = Historical control data; * = p ≤ 0.05 (DUNNETT-test [two-sided])

 

 

Table 2: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding	Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d	HCD (range)
Incomplete ossification of parietal; unchanged cartilage	0.0	1.1	1.1	4.2*	0.2 (0.0 – 1.0)
Splitting of skull bone	1.2	1.2	2.6	8.1**	2.8 (0.0 – 7.1)
Supemumerary 13th rib; cartilage present	3.8	5.3	2.5	13.1*	4.0 (0.0 – 8.7)
Supemumerary 13th rib; cartilage not present	10.7	10.3	7.6	33.5**	6.2 (2.1 – 13.6)
Total fetal skeletal variations	64.8	63.3	66.4	79.3*	63.1 (46.3 – 78.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table 3: Total fetal malformations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 172	25 165	25 176	16 100
Fetal incidence	N (%)	6 (3.5%)	8 (4.8%)	6 (3.4%)	11 (11%)
Litter incidence	N (%)	6 (24%)	6 (24%)	6 (24%)	7 (44%)
Affected fetuses/litter	Mean%	3.9	4.7	3.2	14.3*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided])

 

 

Table 4: Total fetal variations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 172	25 165	25 176	16 100
Fetal incidence	N (%)	122 (71%)	113 (68%)	125 (71%)	82 (82%)
Litter incidence	N (%)	25 (100%)	24 (96 %)	25 (100 %)	16 (100%)
Affected fetuses/litter	Mean%	71.9	69.7	72.5	83.1*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided])

Conclusions:

It is concluded that there is no selective effect of the compound on the respective morphological structures, and the effects would not occur in the absence of maternal toxicity. The compound is no selective teratogen. The no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d.

Executive summary:

In a study performed according to OECD TG 414 and in compliance with GLP, the test substance was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 time-mated female Himalayan rabbits by stomach tube at doses of 100; 300 and 1000 mg/kg body weight/day on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid) in parallel. A standard dose volume of 10 ml/kg body weight was used for each test group. At terminal sacrifice on GD 29, 18-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg body weight/day):

Does:

- Distinct and statistically significant reduction of food consumption (-44%) and gross and net body weight gain(-47% and -54%, respectively)

- Abortions (and subsequent sacrifice) in 7 of 25 animals

- Stomach erosions in 7/25 does, no feces in the small intestine in 5/25 does and watery feces in the intestines in 3/25 high-dose does - Stomach erosions and subsequent massive decrease of individual food consumption particularly affected all does with abortions

- Complete postimplantation loss in 2 individual does (Nos. 76 and 99) secondary to distinct maternal toxicity

Fetuses:

- Statistically significant decrease of mean gravid uterine weights and mean fetal weights

- Slightly higher total fetuses per litter rate of malformations but no specific pattern of malformations, treatment-related adverse fetal findings primarily limited to severely fused sternebrae (bony plate)

- Slightly higher total fetuses per litter rate of variations, namely skeletal variations such as delayed ossification and supernumerary ribs, commonly associated with decreased fetal weight and maternal stress

Test group 2 (300 mg/kg body weight/day):

Does:

- Statistically significant reduction of food consumption (-18%) and net (-37%) body weight gain

Fetuses:

- No biologically relevant differences between treated and control animals

Test group 1 (100 mg/kg body weight/day):

Does:

- No biologically relevant differences between treated and control animals

Fetuses:

- No biologically relevant differences between treated and control animals

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d based on reduced food consumption and body weight gain in the does at 300 mg/kg bw/d and above. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d based on abortions, decreased fetal growth and bone alterations at 1000 mg/kg bw/d. There were no adverse fetal findings evident at a dose not producing maternal toxicity. The compound is no selective teratogen.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study was conducted in accordance with a recognized scientific procedure for determining the developmental toxicity of a test substance when administered repeatedly via inhalation. The study meets national and international scientific standards similar to OECD TG 414 and provides sufficient information to support the conclusions regarding the NOAEL demonstrated from the study data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Fluka Chemie AG (Buchs, Switzerland)
- Batch number of test material: not specified
- Purity: 99.0%

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories, Saint Germain sur l'Arbresle, France
- Age at study initiation: sexually mature and nulliparous females, age further not specified
- Weight at study initiation: 180-200 g
- Housing: singly in polycarbonate cages
- Diet: food pellets, UAR, Alimentation Villemoisson, France
- Water: filtered tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 2 °C
- Humidity: 50 +/- 5 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: not specified

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6±20 m3/h). In order to prevent any leakage of the test atmospheres, the chambers were maintained at a negative pressure of no more than 3-mm water. The chamber temperature was set at 23 +/- 2°C and the relative humidity at 50 +/- 5%. Food and water were withheld during exposures.
The system used for vapor generation consisted in delivering a constant rate of liquid chemical with an infusion pump at the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow , an additional air flow rate was passed through the fritted disk of a heated bubbler containing the test chemical. The vaporized compounds were introduced into the main air-inlet pipe of the exposure chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations were monitored continuously with a gas chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, the exposure levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulide. The resulting samples were then analyzed by gas chromatography using appropriate interval standards.

Details on mating procedure:

- Impregnation procedure: cohaused
- M/F ratio per cage: nulliparous female rats were housed overnight with adult males (1 male: 2 or 3 females) from the same strain and supplier
- Proof of pregnancy: The day that vaginal smears were found to be sperm-positive was considered day 0 of gestation (GD).
- Mated females were housed singly in clear polycarbonate cages with stainless-steel wire lids and hardwood shaving, as bedding.

Duration of treatment / exposure:

days 6 - 20 of gestation

Frequency of treatment:

6 hours per day

Duration of test:

On GD 21, the females were sacrificed

Dose / conc.:

1 200 ppm (nominal)

Remarks:

Analytical concentration: 1206.4 +/- 46.9 (range 1131-1343 ppm)

Dose / conc.:

600 ppm (nominal)

Remarks:

Analytical concentration: 602.3 +/- 38.0 (range 519-685 ppm)

Dose / conc.:

300 ppm (nominal)

Remarks:

Analytical concentration: 301.6 +/- 12.2 (range 276-321 ppm)

Dose / conc.:

100 ppm (nominal)

Remarks:

Analytical concentration: 99.9 +/- 5.0 (range 90-108 ppm)

No. of animals per sex per dose:

22-25 pregnant females per dose

Control animals:

other: yes, concurrently to filtered air

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): Mated females were randomly assigned to treatment groups using a randomization system stratified by body weight on GD0.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GDs 0, 6, 13, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured for the intervals GDs 6-13 and 13-21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of death and live fetuses: Yes

Fetal examinations:

- External examinations: Yes: All live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity
- Soft tissue examinations: Yes: Half of the live fetuses from each lifter were preserved in Bouin's solution and examined for internai soft-tissue changes (Barrow and Taylor, 1969; Wilson, 1965).
- Skeletal examinations: Yes: Half of the live fetuses from each lifte were fixed in ethanol (70%), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination (Staples and Schnell, 1964).
- Head examinations: Not specified

Statistics:

The number of CL, implantation sites, and live fetuses, maternal food consumption and various body weights were analyzed  by ANOVA, followed by Dunnett'st-test. the percentage of non-live  implant, resorptions,and males and the proportion of fetuses with  alterations ineach litter were evaluated by Kruskal-Walles test followed  by Dixon-Massey test. Rates of pregnancy and percentage of litters with  any malformations or external, visceral, or skeletal variations were  analyzed using Fisher's test. Where appropriate, least squares analysis  was performed. The level of significance was p < 0.05.

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No test dam died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Exposure to 300 ppm or higher concentrations of the test substance led to a significant decrease in maternal body weight gain during the first half of exposure. The overall maternal weight gain during GDs 6-21 and the absolute weight gain were significantly less than control at 1200 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in maternal food consumption was observed during the first half of exposure at 300 ppm and higher concentrations (p < 0.05 and p <0.01 at 300 and 1200 ppm, respectively).

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

Endocrine 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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

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:

not examined

Details on maternal toxic effects:

For more detailed information please refer to table 1 in section "Any other information on results incl. tables".

Dose descriptor:

NOAEC

Remarks:

developmental toxicity

Effect level:

1 200 ppm

Based on:

test mat.

Basis for effect level:

other: no maternal developmental toxicity up to and including the highest tested dose

Dose descriptor:

NOAEC

Remarks:

maternal toxicity

Effect level:

300 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight was significantly reduced at 600 ppm (females) and at 1200 ppm (all, male and female fetuses). These decreases amounted to 5% of the concurrent control values at 1200 ppm.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences between the control and treated groups in the incidences of either individual or total external and visceral variations, or of individual skeletal variations.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences between the control and treated groups in the incidences of either individual or total external and visceral variations, or of individual skeletal variations. The only statistically significant changes were higher mean percentages of fetuses with skeletal variations or any variations at the highest concentration of the test substance, compared with the concurrent control. The biological relevance of these findings is limited by the fact that the observed incidences occurred with no clear concentration-related pattern. These increases might be considered at most as slight fetotoxicity.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Occasional visceral malformations occurred in low frequency and were distributed across the different groups, including control.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

For more detailed information please refer to tables 2 and 3 in section "Any other information on results incl. tables".

Dose descriptor:

NOAEC

Remarks:

developmental toxicity

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal variations

Dose descriptor:

NOEC

Remarks:

fetotoxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

TABLE 1: Change in Weight During Gestation in Sprague-Dawley Rats Inhaling the test substance on Days 6 to 20 of Gestation and Euthanized on Day 21.

 

Concentration ppm/6 h/day	No. of dams^a	Body weight (g) on GD 6	Body weightgain (g) on GD			Absolute weight gain (g)^b
			6±13	13±21	6±21
0	24	257 ± 15	33 ± 6	100± 16	133 ± 19	27 ± 10
100	24	259 ± 16	31 ± 8	100± 15	132 ± 18	26 ± 8
300	25	258 ± 15	27 ±6**	106± 12	132 ± 14	25 ± 9
600	22	264 ± 18	26 ± 5**	102± 19	128 ± 19	21 ± 10
1200	25	261 ± 18	21 ± 6**	98 ± 13	119 ± 15*	19 ± 9**

Note.Values are expressed as means ± SD.

^a Includes all dams pregnant at euthanization.

^bDay 21 body weight) 2 (gravid uterus weight) 2 (Day 6 body weight).

*,** Denote significant differences from the control (0 ppm) value , p, 0.05 and p , 0.01, respectively.

 

TABLE 2: Reproductive Parameters in Sprague-Dawley Rats Inhaling the test substance on Days 6 to 20 of Gestation and Euthanized on Day 21.

 

Concentration (ppm/6 h/day)

Test animals (dams)

Litters with implants

Litters with live fetuses

No. dead/ no. treated

% of females pregnant at euthanization

No. of litters

No. of corpora lutes per dam

No. of implantation sites per litter

% of nonlive implants per litter^a

% of resorption sites per litter

No. of litters

No. of live fetuses per litter

% of males per litter

Average fetal body weight (g) per litter

All

Males

Females

0

0/26

92.3

24

15.63 ± 1.69

14.63 ±2.43

7.12 ± 12.12

6.52 ± 9.66

24

13.75 ± 3.01

53.40 ± 16.29

5.70±0.26

5.84 ± 0.29

5.54 ± 0.25

100

0/26

92.3

24

15.75 ± 1.45

14.71 ± 1.90

4.00 ± 6.80

4.00 ± 6.80

24

14.13 ± 2.11

51.50 ± 15.16

5.59± 0.25

5.71 ± 0.28

5.47 ± 0.27

300

0/26

96.2

25

16.12 ± 1.56

15.24 ±   1.59

4.05 ± 3.89

4.05 ± 3.89

25

14.60 ± 1.38

52.83 ± 12.70

5.53± 0.24

5.67 ± 0.26

5.38 ± 0.20

600

0/26

84.6

22

16.27 ± 1.52

15.18 ±2.54

5.28 ±8.08

5.28 ± 8.08

22

14.50 ± 3.02

46.59 ± 11.86

5.51± 0.33

5.72 ± 0.36

5.33 ± 0.35*

1200

0/27

92.6

25

16.76 ± 3.30

15.08 ± 2.00

6.27 ± 6.08

6.27 ± 6.08

25

14.12 ± 1.99

46.22 ± 15.16

5.40± 0.24**

5.56 ± 0.29**

5.26 ± 0.24**

^aResorptions plus dead fetuses.

Values are expressed as means±SD.

*,** denote significant differences from the control (0 ppm) value,p , 0.05 and p , 0.01, respectively.

 

TABLE 3: Incidence of Malformations and Variations in Fetuses of Sprague Dawley Rats Inhaling n-Butyl Methacrylate on Days 6 to 20 of Gestation.

 

Concentration (ppm/6 h/day)	0	100	300	600	1200
Total no. fetuses (litters) examined^a External Visceral Skeletal	330 (24) 165 (24) 165 (24)	339 (24) 170 (24) 169 (24)	365 (25) 183 (25) 182 (25)	319 (22) 160 (22) 159 (22)	353 (25) 177 (25) 176 (25)
Malformations Aorta transposed and pulmonary artery stenosis Diaphragmatic hernia No. (%) fetuses with any malformations No. (%) litters with any malformations Mean % fetuses with any malformations per lifter^b	0 1 (1) 1 (0.3) 1 (4.2) 0.28 ± 1.36	0 0 0 0 0	0 2 (2) 2 (0.5) 2 (8.0) 0.70 ± 2.41	0 0 0 0 0	1 (1) 0 1 (0.3) 1 (4.0) 0.29 ± 1.43
External variations Club foot, unilateral No. (%) fetuses with external variations No. (%) litters with external variations Mean % fetuses with external variations per lifter^b	0 0 0 0	1 (1) 1 (0.3) 1 (4.2) 0.30 6 1.46	0 0 0 0	1 (1) 1 (0.3) 1 (4.5) 0.27 ± 1.25	1 (1) 1 (0.3) 1 (4.0) 0.30 ± 1.43
Visceral variations Dilated renal pelvis Distended ureter No. (%) fetuses with visceral variations No. (%) litters with visceral variations Mean (%) fetuses with visceral variations per litter^b	1 (1) 6 (6) 6 (3.6) 6 (25.0) 3.37 ± 6.02	1 (1) 20 (8) 20 (11.8) 8 (33.3) 11.07 ± 19.48	2 (1) 10 (6) 12 (6.6) 7 (28.0) 6.95 ± 15.13	0 7 (6) 7 (4.4) 6 (27.3) 4.25 ± 7.49	0 19 (9) 19 (10.7) 9 (36.0) 10.57 ± 17.27
Skeletal variations
Skull Hyoid, incomplete ossification or unossified^c Supraoccipital, incomplete ossification	0 0	1 (1) 1 (1)	1 (1) 0	0 0	1 (1) 0
Sternebrae incomplete ossification or unossi®ed second or fourth asymmetric/bipartite asymmetric, severe extra ossification site between fifth and sixth sternebrae	14 (7) 1 (1) 1 (1) 0	8 (5) 0 0 0	7 (6) 1 (1) 1 (1) 1 (1)	5 (4) 0 0 0	26 (12) 0 1 (1) 0
Rib(s) cervical, rudimentary 14th, supernumerary 13th, short 13th, right, short and 13th left, unossified floating	4 (3) 4 (4) 1 (1) 1 (1) 1 (1)	4 (3) 4 (4) 1 (1) 0 0	0 14 (9) 0 0 0	2 (2) 5 (5) 0 0 0	7 (7) 2 (2) 2 (2) 0 0
Thoracic vertebral centra, incomplete ossification (one or two)	1 (1)	6 (5)	9 (6)	2 (1)	13 (8)
Lumbar vertebral centra, incomplete ossification (one)	0	0	0	1 (1)	0
No. (%) fetuses with skeletal variations	23 (13.9)	23 (13.6)	30 (16.5)	12 (7.5)	43 (24.4)
No. (%) litters with skeletal variations	12 (50.0)	15 (62.5)	16 (64.0)	9 (40.9)	18 (72.0)
Mean (%) fetuses with skeletal variations per litter^b	13.36 ± 18.26	13.20 ± 12.60	16.36 ± 15.09	7.57 ± 12.01	24.54 ± 23.58*
No. (%) fetuses with any variations	29 (8.8)	44 (13.0)	42 (11.5)	20 (6.3)	63 (17.8)
No. (%) litters with any variations	14 (58.3)	18 (75.0)	17 (68.0)	10 (45.4)	21 (84.0)
Mean % fetuses with any variations per litter^b	8.40 ± 10.61	12.37 ± 12.65	11.51 ±12.30	6.13 ± 8.34	17.88 ± 15.25**

^aThe incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined.

^bMean 6 SD.

^cUnossifed, alizarine red S negative.

* and ** denote significant difference from control (air),p , 0.05 and p , 0.01, Dixon-Massey's test.

Conclusions:

Inhalation exposure of rats to the test substance did not produce evidence of embryolethality or teratogenicity up to concentrations which produced overt maternal toxicity.

Executive summary:

In a study comparable to the OECD TG 414, groups of 22-25 pregnant female rats were given whole-body inhalation exposures to the test substance at target concentrations of 0, 100, 300, 600 or 1200 ppm (analytical concentrations: 0, 99.6 +/- 5.0, 301.6 +/- 12.2, 602.3 +/-38.0, 1206.4 +/- 46.9 ppm) for 6 hr/day, during days 6 to 20 of gestation (GD). Maternal toxicity (decreased body weight gain) was shown at 300 to 1200 ppm. Feed consumption was decreased at 1200 ppm. No dam died during the test and there were no adverse effects on the average number of implantations and live fetuses, incidence of non-live fetuses, or on resorptions. Fetal body weights of male pups were significantly reduced at 1200 ppm, and females at 600 and 1200 ppm test substance. There were no significant differences between control and treated groups for external, visceral, or skeletal malformations. A significant increase in the skeletal variations per litter occurred at 1200 ppm test substance, compared to controls. The biological relevance of these findings is limited by the factthat the observed incidences occurred with no clear concentration-related pattern. These increases might be considered atmost as slight fetotoxicity. The concluded NOAEC for developmental toxicity was 600 ppm. There was no evidence of embryolethality or teratogenicity with the test substance. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP and guideline compliant study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 012 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable study similar to guideline

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable studies in rats (inhalativ) and rabbits (oral) for an analogous substance were used to assess the potential of the test item for developmental toxicity.

Oral

In a study performed according to OECD guideline 414 and in compliance with GLP (reliability 1, 2009), the structural analogue was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 time-mated female Himalayan rabbits by stomach tube at doses of 100; 300 and 1000 mg/kg body weight/day on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid) in parallel. A standard dose volume of 10 ml/kg body weight was used for each test group. At terminal sacrifice on GD 29, 18-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

1000 mg/kg bw/d:

Does:

- Distinct and statistically significant reduction of food consumption (-44%) and gross and net body weight gain(-47% and -54%, respectively)

- Abortions (and subsequent sacrifice) in 7 of 25 animals

- Stomach erosions in 7/25 does, no feces in the small intestine in 5/25 does and watery feces in the intestines in 3/25 high-dose does - Stomach erosions and subsequent massive decrease of individual food consumption particularly affected all does with abortions

- Complete postimplantation loss in 2 individual does (Nos. 76 and 99) secondary to distinct maternal toxicity

Fetuses:

- Statistically significant decrease of mean gravid uterine weights and mean fetal weights

- Slightly higher total fetuses per litter rate of malformations but no specific pattern of malformations, treatment-related adverse fetal findings primarily limited to severely fused sternebrae (bony plate)

- Slightly higher total fetuses per litter rate of variations, namely skeletal variations such as delayed ossification and supernumerary ribs, commonly associated with decreased fetal weight and maternal stress

300 mg/kg bw/d:

Does:

- Statistically significant reduction of food consumption (-18%) and net (-37%) body weight gain

Fetuses:

- No biologically relevant differences between treated and control animals

100 mg/kg bw/d:

Does:

- No biologically relevant differences between treated and control animals

Fetuses:

- No biologically relevant differences between treated and control animals

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d based on reduced food consumption and body weight gain in the does at 300 mg/kg bw/d and above. The NOAEL for prenatal developmental toxicity is 300 mg/kg bw/d based on abortions, decreased fetal growth and bone alterations at 1000 mg/kg bw/d. There were no adverse fetal findings evident at a dose not producing maternal toxicity. The compound is no selective teratogen.

In another supporting study realiable screening study according to OECD 421 (reliability 2, 2008) with the target substance was administered at 0 (control group: aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100, 300 and 1000 mg/kg to Sprague- Dawley SPF rats, for 14 days before mating and throughout the mating period until the day before autopsy in males (42 days), and for 14 days before mating and throughout the mating period and gestation period until day 4 of nursing in females (42-48 days), and the repeated dose toxicity and reproductive and developmental toxicity were investigated according to a OECD TG 421 study. In the parent animals, test substance administration had no observable effect on estrous cycle, days required until copulation, copulation index, insemination index or conception index. Furthermore, test substance administration had no observable effect on delivery index, gestation period, number of corpora lutea, number of implantation marks, implantation index, stillbirth index, number of liveborn pups, livebirth index or sex ratio, and no abnormalities in nursing condition were observed. Therefore, test substance administration was thought to have had no effect on reproductive functions such as copulation ability in males or females, or on insemination ability or conception ability, or on pregnancy maintenance, delivery and nursing behavior, etc. of dams, even in the 1000 mg/kg group. In the liveborn pups, no changes due to test substance administration were observed in body weight at birth or on day 4 of nursing, in males or females, and no changes due to test substance administration were observed on external examination at birth, or in the autopsy findings on day 4 of nursing, or in the viability index. It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance in females was less than 100 mg/kg/day, and that the no-observed effect dose with respect to reproduction and development toxicity in female parents and pups was 1000 mg/kg/day.

 

Inhalation

In a study comparable to the OECD TG 414 (reliability 2, 1999), groups of 22-25 pregnant female rats were given whole-body inhalation exposures to the structural analogue at target concentrations of 0, 100, 300, 600 or 1200 ppm (analytical concentrations: 0, 99.6 +/- 5.0, 301.6 +/- 12.2, 602.3 +/-38.0, 1206.4 +/- 46.9 ppm) for 6 h/day, during days 6 to 20 of gestation (GD). Maternal toxicity (decreased body weight gain) was shown at 300 to 1200 ppm. Feed consumption was decreased at 1200 ppm. No dam died during the test and there were no adverse effects on the average number of implantations and live fetuses, incidence of non-live fetuses, or on resorptions. Fetal body weights of male pups were significantly reduced at 1200 ppm, and females at 600 and 1200 ppm test substance. There were no significant differences between control and treated groups for external, visceral, or skeletal malformations. A significant increase in the skeletal variations per litter occurred at 1200 ppm test substance, compared to controls. The biological relevance of these findings is limited by the factthat the observed incidences occurred with no clear concentration-related pattern. These increases might be considered at most as slight fetotoxicity. The concluded NOAEC for developmental toxicity was 600 ppm. There was no evidence of embryolethality or teratogenicity with the test substance. 

In another supporting study the structural analogue Methyl Methacrylate was tested for its prenatal developmental toxicity after oral application in Himalayan rabbits according to OECD TG 414 in compliance with GLP (BASF SE, 2009). The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50, 150 and 450 mg/kg body weight/day on GD 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

In the mid and high dose group, reduced food consumption (-18% and -13%, resp.) and body weight gain (-31% and -27%, resp.) were noted. No test substance-related adverse effects were observed on gestational parameters or fetuses. In the low dose group, no test substance-related adverse effects on does, gestational parameters or fetuses were observed.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is ≥ 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose

In a developmental toxicity study according to OECD 414 and conducted in compliance with GLP standards (Solomon et al. 1991), methyl methacrylate (99.9% active ingredient) was administered by inhalation exposure to 5 groups (27 rats/group) of presumed pregnant rats (Crl:CDBR) at concentrations of ca. 0 (control), 0.4, 1.2, 4.8, 8.3 mg/L (corresponding to 99, 304, 1178, and 2028 ppm) for 6 hr/day on days 6-15 of gestation (GD) . All doses were administered by a whole-body inhalation exposure under dynamic conditions. Clinical signs were recorded daily on GD 0-20. The dams were weighed on GD 0, 6, 8, 10, 13, 16 and 20. Feed consumption was recorded during gestation. On GD 20, the dams were euthanized and the thoracic and abdominal cavities were examined for gross changes. Each uterus was weighed and corpora lutea, implantation sites and resorptions were counted. Fetuses were weighed, sexed, examined for external alterations and one-half of the fetuses from each litter were examined for visceral alterations.

No treatment-related deaths were noted at any concentration tested. The only clinical sign noted was a minimal increase in the incidence of scant feces at ca. 8.3 mg/L. At all exposure levels tested losses in maternal body weight or decreases in maternal body weight gain and decreases in maternal feed consumption were noted. Loss in maternal body weight during the first two days of exposure followed by an overall reduced increase in maternal body weight gain during the treatment period was detected for the 4.8 mg/L and 8.3 mg/L groups. Slight effects were observed for the 0.4 and 1.2 mg/L treatment groups as indicated by a transiently (during the first two days of exposure) reduced maternal body weight gain. According to the authors, a maternal no observed effect level (NOEL) could therefore not be demonstrated. No embryo of fetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 8.3 mg/L. Therefore toxicity to the conceptus was not evident even at exposure levels that resulted in overt maternal toxicity.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available studies performed with the structural analogue as well as the test item itself are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.

.