5-bromo-1,3-dichloro-2-fluorobenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 25 June 2020, Experimental end: 27 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-1,3-dichloro-2-fluorobenzene

EC Number:

640-454-2

Cas Number:

17318-08-0

Molecular formula:

C6H2 BrCl2F

IUPAC Name:

5-bromo-1,3-dichloro-2-fluorobenzene

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Supplier: Charles River (UK) Limited, Margate, Kent, United Kingdom
Sex: time-mated females
Age: 9 - 10 weeks of age on arrival
Acclimatisation: at least two days
Weight: 173 to 250 g
Housing: solid-floor cages (RB3 supplied by North Kent Plastics, NKP), with bedding provided. Bedding was changed regularly to maintain hygiene.
Temperature: 19 to 23 °C
Humidity: 40 to 70%
Illumination: 12 hours light to 12 hours darkness cycle
Diet: pelleted rodent diet, VRF 1 manufactured by SDS and supplied by Charles River (UK) Limited, ad libitum
Water: mains tap water, ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was inspected for any signs of crystallisation. The required quantity of test item was added to a container, together with a magnetic stirring bar. Approximately 60% of the final required quantity of vehicle (or the total quantity of vehicle for formulations prepared by weight) was added and the mixture was stirred until the test item was suitably dispersed. For formulations prepared by volume, the formulation was made up to final quantity with the remaining vehicle and stirred again until suitably homogeneous.
Formulations were divided into daily aliquots and stored at room temperature in amber glass bottles. They were stirred from 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity, which was checked visually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from all formulations. Sets of samples (for analysis or for contingency) were taken from each test item formulation prepared for the first day of dosing and were analysed for test substance using a validated method to confirm homogeneity and also achieved concentrations. Having satisfactorily confirmed achieved concentrations and homogeneity on the first day of dosing, the samples for towards the end of the dosing period were analysed simply to confirm satisfactory concentration. For Controls, sets of samples (for analysis or for contingency) were taken from the vehicle on the first day of dosing and on one occasion towards the end of the dosing period and were analysed to confirm absence of test item.
The individual measured concentrations of the test substance were within 8% of their nominal values. The analysis of formulations at the start of the study also confirmed that the formulations were homogeneous as shown by a relative standard deviation (% RSD) of 1.1% or lower. These data thereby fulfilled the acceptance criteria (±10% of nominal for achieved concentration and a % RSD no greater than 5% for homogeneity).

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

Dosing by oral gavage from Day 6 to 19 of gestation, inclusive

Frequency of treatment:

Daily

Duration of test:

The animals were killed on Day 20 of gestation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected on the basis of results from a 14-day non-pregnant dose range-finding study, a developmental toxicity screening study (OECD TG 422) and the preliminary results of an on-going 90-day study (OECD TG 408). In the screening study, females in the 300 mg/kg/day dose group had to be euthanised by Day 9 due to general poor physical condition and body weight loss. Females given 150 mg/kg/day also showed significantly lower body weight gain in the first two weeks of the treatment period compared with the Control group. In the 90-day study, in the first half of the treatment period no test item related clinical signs or effects on the body weight parameters were observed at any dose level up to 50 mg/kg/day. Based on the female mortality and the reduced body weight gain, 100 mg/kg/day was selected as the top dose for this study. The subsequent doses of 30 mg/kg/day and 10 mg/kg/day were calculated by a factor of approximately three.
Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on Day 0 of gestation (making sure that females mated with the same male were spread across the groups) by the supplier. The cages were positioned in the battery using a randomised cage allocation procedure, starting at the top left-hand corner of the rack and then working from left to right, top to bottom. All groups were allocated to each rack.

Examinations

Maternal examinations:

Clinical observations: animals were examined twice daily for mortality and morbidity and given a detailed clinical examination daily. Animals were also observed circa one to two hours after dosing.
Body weight: the weights were recorded on Day 0 of gestation, and daily from Day 5 to 20 of gestation
Food intake: food amounts consumed by each animal were recorded over Days 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation
At the termination of the studies, the animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative.
The thyroids and liver from all dams were weighed after trimming of fat and other
contiguous tissue. Thyroids were weighed together after fixation.
For all dams, the liver, thyroids (including parathyroids) and any gross lesions (including abnormal teeth) were preserved in 10% buffered formalin.
For all animals, the thyroids (and abnormal teeth from dams with tooth abnormalities) were wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically. To aid interpretation of apparent liver weight increases, the liver from all pregnant females was wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically.

Ovaries and uterine content:

The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70% IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in 10% buffered formalin.
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses.
The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The foetuses and their placentae were removed and the uterus and ovaries were retained in 10% buffered formalin.

Blood sampling:

Thyroid hormone assessment: Blood samples (0.2 mL) were taken on the day of necropsy from the tail vein of all animals into plain tubes (a gel separator tube without anticoagulant). All samples were taken between 08:00 and 08:42 hours and the animals were sampled in a random cage and group order. Blood samples were allowed to clot for at least 30 minutes at room temperature before being centrifuged (3000 g, 10 minutes, at approximately 4 °C). The resulting serum was aliquoted into two tubes before being stored frozen (< -70 °C) until analysis.
One aliquot of the samples was analysed for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) using validated methods.

Fetal examinations:

On Day 20 of gestation, live foetuses were killed by rapid cooling, weighed, sexed and examined for external abnormalities. Anogenital distance was measured using digital callipers, measuring between the anus and caudal end of the genital tubercle.
All live foetuses were briefly placed in 70% IDA and subjected to micro-dissection, where the viscera were examined (paying particular attention to the reproductive tract for signs of altered development) and the foetuses eviscerated. Approximately 50% of the live foetuses were allocated to the fixed head examination where they were decapitated and the heads were fixed in Bouin's fluid and examined by serial sectioning. A coronal section was made through the head of the intact foetuses along the frontal parietal suture and the brain examined.
All carcasses were transferred to 95% IDA and cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.

Statistics:

Information on statistics is provided under "Any other information on materials and methods"

Indices:

Pre-implantation loss (%) is calculated as (number of corpora lutea - number of implantation sites) divided by number of corpora lutea multiplied by 100
Post-implantation loss (%) is calculated as (number of implantation sites - number of live foetuses) divided by number of implantation sites multiplied by 100
Anogenital distance index (ACGI) is calculated as the ratio of Anogenital distance (mm) to cubed root body weight (g)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, there was an initial, dose-related, reduction in group mean body weight gain compared with Controls, with some animals given 100 mg/kg/day losing weight over Days 6 to 9 of gestation. After this point, there was an increase in body weight, however, overall group mean body weight gain in the groups given 30 or 100 mg/kg/day was statistically significantly (p ≤ 0.01) lower than Controls. This resulted in group mean body weight on Day 20 of gestation being 5% and 6% lower than Controls, respectively.
Terminal body weight adjusted for the weight of the gravid uterus was statistically
significantly (p ≤ 0.05) lower (7%) than Controls for females given 30 or 100 mg/kg/day.
Group mean body weight, body weight gain and terminal body weight adjusted for the
weight of the gravid uterus were similar to Controls for females given 10 mg/kg/day.
However, when the terminal body weight gain was adjusted for the weight of the uterus it was 33% lower than Controls (p≤0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food intake for females given 30 or 100 mg/g/day was statistically significantly (p ≤ 0.01) lower than Controls throughout the dosing period, with an overall food intake 12% and 18% lower than Controls, respectively

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, there were 19% and 37%, respectively, decreases in group mean T4 concentrations (p≤0.01). The group mean T4 concentrations were outside the limited historical Control data range available for thyroid hormone concentrations on Day 20 of gestation.
Although the mean total T4 concentrations at 30 and 100 mg/kg/day were lower than those in the controls on day 20 of gestation and below the limited historical Control ranges, there were no corresponding changes to either total T3 or TSH concentrations. Given the inherent individual variability in hormone concentrations and since there were no effects on thyroid weights or microscopic changes at these dose levels, it is considered that the lowering in mean total T4 seen in this study was not adverse.

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):

At 100 mg/kg/day, group mean body weight adjusted liver weights were higher than the
Controls (up to 12% higher; p≤0.01). Most individual absolute and body weight related values were outside the Control range. These changes did not correlate with the microscopic findings in the liver.
Group mean absolute liver weight was statistically significantly lower (p≤0.05 to p≤0.01) than Controls for females given 10 or 30 mg/kg/day, however, the mean adjusted liver weights and body weight-related liver weights were similar to Controls. Therefore, this slight lowering in absolute liver weight was considered not to be test item related. There was no effect on absolute or body weight adjusted thyroid weight at any dose level.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal white teeth were noted in one Control and one female given 30 mg/kg/day at
necropsy, for which there was no histopathological correlate. There were no test item-related macroscopic abnormalities noted in the liver or thyroid glands at necropsy. The observed findings were generally consistent with changes encountered in rats of this age and strain kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the liver of females given 100 mg/kg/day. These included occasional necrotic hepatocytes (minimal) in 13 out of 22 females and an increased incidence of focal/multifocal inflammatory cell infiltrates (minimal), most of which were adjacent to the necrotic hepatocytes.
There were no microscopic findings to account for the increased liver weights in animals given 100 mg/kg/day.
Also, there were no microscopic findings to account for the abnormal white teeth noted in two females at necropsy. There were no test item-related microscopic findings in the thyroid glands examined in females given 10, 30 or 100 mg/kg/day of the test item.
Other observed findings were generally consistent with changes encountered in rats of this age and strain kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

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:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

On Day 20 of gestation, there were 21, 20, 22 and 20 females with live foetuses in the Control group and groups given 10, 30 or 100 mg/kg/day, respectively. There was no effect on the uterine or implantation data at any dose level, with similar incidences of pre- and post-implantation loss and comparable numbers of live foetuses per litter across the groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, the group mean foetal weights for male and female foetuses
combined, or separately, were slightly below the background data range, although only 3% lower than the Control group mean and there was no clear dose relationship.

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:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant decrease in group mean anogenital distance index (AGDI) for male foetuses given 10, 30 and 100 mg/kg/day compared with Control (6%, 11% or 10%, respectively; p≤0.05 to p≤0.01). While this was not dose-related, the group mean values were below the limited historical Control data for all groups
given the substance.
There was a slight, dose-related increase in group mean AGDI for female foetuses compared with Control (4%, 7% or 10%, respectively) achieving statistical significance at 30 and 100 mg/kg/day (p≤0.05; p≤0.01). The group mean values were within the limited historical Control data, therefore the apparent intergroup differences were considered to reflect normal biological variation and not to be associated with treatment.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Skeletal malformations:

effects observed, treatment-related

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Major foetal abnormalities were observed in five foetuses; one foetus in the Control group and in each of the groups given 30 or 100 mg/kg/day and in two foetuses from two litters in the group given 10 mg/kg/day. Details of the abnormalities were as follows:
- control group, dam 17, foetus R4: fused jugal and maxilla
- 10 mg/kg/day group, dam 30, foetus R3: malformed cervical vertebral neural arch(es)
- 10 mg/kg/day group, dam 37, foetus R6: microphthalmia
- 30 mg/kg/day group, dam 49, foetus L4: dome shaped head, cheiloschisis, cleft palate, anophthalmia, malformed premaxilla, orbital cavity reduced in size, malformed tympanic ring
- 100 mg/kg/day group, dam 75, foetus R3: incomplete interventricular septum
The nature, incidence and intergroup distribution of these major foetal abnormalities were considered to be incidental and not test item-related.

At 30 and 100 mg/kg/day, there were statistically significant higher numbers of litters with foetuses with the minor abnormality of oedema of the ventral surface of the body (p≤0.01) and the incidences were dose-related. In addition, oedema of the ventral region of the thorax was noted in foetuses from two litters in each of these groups and at 100 mg/kg/day, oedema of the ventral region of the neck was seen in foetuses from two litters and protruding tip of the tongue in foetuses from seven litters. None of these minor abnormalities has been noted in the historical Control data and were considered to be adverse.
At 100 mg/kg/day, there was a higher number of litters with foetuses with minor and variant abnormalities of the cervical vertebrae including mishapen neural arch(es), fused cartilaginous ventral plate and additional cartilaginous ventral plate, and cartilaginous additional cartilaginous ventral plate on the 5th cervical vertebra. In addition, there was an increase in the number of litters with foetuses showing the minor abnormality of nonossification of the caudal vertebral neural arches when compared with Controls. All values were outside the historical Control ranges.
Other minor and variant foetal abnormalities that attained statistical significance at 100 mg/kg/day when compared with the Controls, included partially fused jugal and maxilla, uni- or bilateral cervical rib, one or more misshapen or misaligned sternebrae, incomplete ossification of one or more metatarsals, increased pelvic cavitation, interrupted costal cartilage and non, or incomplete, ossification of the 5th sternebra. The incidences of these abnormalities were low, affecting a small number of litters and generally remained within the historical Control data ranges.
The incidences of the minor and variant foetal abnormalities that attained statistical significance when compared with the Controls are presented in a separate table.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Remarks on result:

other: There was a reduction of anogenital distance in male foetuses at all dose levels

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: trunk

external: anogenital distance

external: thorax

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

presumably yes

Any other information on results incl. tables

Table 1: Minor abnormalities

Finding		Dose level (mg/kg/day)				Background data range
		0	10	30	100
Oral cavity: - protruding tongue tip	# foetuses	0	0	0	13 (6.2)JJ	Not seen
	# litters	0	0	0	7CC
Neck: - oedema in ventral region	# foetuses	0	0	0	18 (7.9)	Not seen
	# litters	0	0	0	2
Thorax: - oedema in ventral region	# foetuses	0	0	3 (5.5)	11 (5.0)J	Not seen
	# litters	0	0	2	2
Body: - oedema in ventral region	# foetuses	0	0	112 (52.6)JJ	137 (68.6)JJ	Not seen
	# litters	0	0	12CC	17CC
Skull: - jugal and maxilla: uni- or bilateral partial fusion	# foetuses	0	0	0	3 (3.1)J	1 to 4 foetuses, 0.6 to 3.1% of litters, 1 to 2 litters
	# litters	0	0	0	3C
Cervical vertebra: - one or more neural arch: misshapen	# foetuses	2 (1.0)	11 (5.4)	8 (7.5)	17 (8.9)JJ	1 to 7 foetuses, 0.5 to 4.1% of litters, 1 to 6 litters
	# litters	2	6	6	14CC
Cervical vertebra: - cartilaginous ventral plate: uni- or bilateral: fused	# foetuses	1 (0.4)	11 (6.2)	5 (2.2)	13 (6.7)JJ	1 to 4 foetuses, 0.3 to 2.5% of litters, 1 to 4 litters
	# litters	1	8	5	11CC
Cervical vertebra: - additional cartilaginous ventral plate: uni- or bilateral: fused	# foetuses	1 (0.4)	11 (6.2)	5 (2.2)	14 (7.1)JJ	1 to 4 foetuses, 0.4 to 2.5% of litters, 1 to 4 litters
	# litters	1	8	5	12CC
Caudal vertebra: - number of neural arches: 0 ossified	# foetuses	1 (0.5)	3 (1.3)	5 (2.2)	9 (4.5)J	1 to 3 foetuses, 0.4 to 3.7% of litters, 1 to 3 litters
	# litters	1	3	3	5
Rib: - uni-bilateral: cervical	# foetuses	4 (1.7)	8 (4.4)	2 (0.8)	12 (6.3)	1 to 13 foetuses, 1.0 to 12.6% of litters, 1 to 9 litters
	# litters	3	6	2	8C
Sternum: - one or more sternebra: misshapen or misaligned	# foetuses	0	1 (0.5)	5 (2.1)J	3 (1.3)J	1 to 13 foetuses, 0.7 to 5.7% of litters, 1 to 10 litters
	# litters	0	1	5	2
Hindlimb: - one or more metatarsal: incomplete ossification	# foetuses	3 (1.5)	5 (2.3)	9 (3.7)	22 (10.6)J	1 to 9 foetuses, 0.4 to 4.2% of litters, 1 to 7 litters
	# litters	3	4	5	9C

Figures in brackets are group mean percentage values; J = p<0.05, JJ = p<0.01 (Jonckheere Trend Test); C = p<0.05, CC = p<0.01 (Cochran-Armitage Test)

Table 2: Variations

Finding		Dose level (mg/kg/day)				Background data range
		0	10	30	100
Abdominal cavity: - kidney, uni- or bilateral: increased pelvic cavitation	# foetuses	0	0	1 (0.4)	3 (1.7)J	1 to 5 foetuses, 0.5 to 5.4% of litters, 1 to 3 litters
	# litters	0	0	1	2
Cervical vertebra: - ventral tubercle: not ossified	# foetuses	63 (31.3)	102 (45.3)J	113 (51.2)JJ	131 (61.6)JJ	26 to 107 foetuses, 24.3 to 52.4% of litters, 12 to 21 litters
	# litters	18	19	21	20C
Cervical vertebra: - cartilaginous ventral plate: uni- or bilateral : on 5th cervical vertebra	# foetuses	2 (1.0)	10 (4.9)	7 (6.9)	15 (8.0)JJ	1 to 9 foetuses, 0.4 to 5.9% of litters, 1 to 6 litters
	# litters	2	6	5	13CC
Cervical vertebra: - additional cart ventral plate: uni- or bilateral: on 5th cervical vertebra	# foetuses	1 (0.4)	11 (6.2)	4 (1.6)	17 (8.5)JJ	1 to 6 foetuses, 0.3 to 2.7% of litters, 1 to 5 litters
	# litters	1	8	4	14CC
Rib: - one or more: costal cartilage interrupted	# foetuses	75 (36.8)	86 (40.7)	116 (52.0)J	101 (49.6)J	1 to 108 foetuses, 0.7 to 50.5% of litters, 1 to 22 litters
	# litters	20	20	22	20
Sternum: - 5th sternebra: not ossified	# foetuses	7 (3.4)	13 (6.0)	11 (5.0)	33 (15.8)JJ	1 to 13 foetuses, 0.4 to 10.7% of litters, 1 to 7 litters
	# litters	3	9	6	12CC
Sternum: - 5th sternebra: incomplete ossification	# foetuses	18 (8.8)	22 (9.9)	36 (15.0)	42 (19.4)J	1 to 20 foetuses, 1 to 15.3% of litters, 1 to 11 litters
	# litters	11	10	15	15C
Forelimb: - one or more digits – bilateral: phalanges ossified	# foetuses	29 (12.2)	40 (18.6)	49 (26.0)	56 (29.1)J	15 to 75 foetuses, 9.4 to 45.4% of litters 9 to 15 litters
	# litters	9	7	15	12

Figures in brackets are group mean percentage values; J = p<0.05, JJ = p<0.01 (Jonckheere Trend Test); C = p<0.05, CC = p<0.01 (Cochran-Armitage Test)

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal toxicity and embryo-foetal development was considered to be 10 mg/kg/day.

Executive summary:

The developmental toxicity of the substance was studied under GLP to OECD TG 414. Four groups of 22 sexually mature, time-mated female Crl:WI(Han) rats were given the test substance in corn oil by gavage at zero (vehicle control), 10, 30 or 100 mg/kg bw/day, once daily, at a dose volume of 2 mL/kg from Day 16 to 19 of gestation, inclusive. There were no premature deaths and no abnormal clinical signs of maternal toxicity. There was a dose-related reduction in maternal body weight and food consumption at 30 or 100 mg/kg bw/day. At these doses, there was a statistically significant decrease in group mean T4 concentrations (19% and 37%, respectively) compared to the control animals. However, there was no effect on T3 and TSH concentrations at 30 or 100 mg/kg bw/day. Furthermore, there was no effect on thyroid weights or microscopic findings, and thus the decreased T4 concentrations were considered to be not adverse. At 100 mg/kg bw/day only, group mean absolute and body weight-related liver weights were up to 12% higher than that of the controls. Macroscopic findings of abnormal white teeth were noted in one control and in one female given 30 mg/kg bw/day, for which there was no histopathological correlate. Substance-related microscopic minimal changes of occasional necrotic hepatocytes were observed in the liver of 13 out of 22 females given 100 mg/kg bw/day. Furthermore, there was an increased incidence of focal/multifocal inflammatory cell infiltrate (minimal) at this dose level. On day 20 of gestation, there were 21, 20, 22 and 20 females with live foetuses in the control group and the groups given 10, 30 or 100 mg/kg bw/day, respectively. There was no effect on the uterine or implantation data, foetal weight, placental weight or the mean sex ratio at any dose level that was considered to be associated with the test substance. The group mean anogenital distance index (AGDI) for male foetuses at 10, 30 and 1000 mg/kg bw/day was significantly lower than that of controls (6%, 11% or 10%, respectively). There were no major foetal abnormalities that were attributed to the test substance. At 30 or 100 mg/kg bw/day there was a higher number of litters with foetuses that had the minor foetal abnormality of oedema. Furthermore, at 100 mg/kg bw/day there was a higher number of litters with foetuses with a protruding tip of the tongue and minor or variant abnormalities of the cervical vertebrae. 

In conclusion, the administration of the substance to pregnant Crl:WI(Han) rats once daily by oral gavage from days 6 to 19 of gestation, inclusive, was well tolerated at dose levels of 10, 30 or 100 mg/kg bw/day. Signs of maternal toxicity were limited to 30 or 100 mg/kg bw/day. There was no effect of the substance on pregnancy or foetal growth. However, there was an increase in the incidences of minor foetal abnormalities at 30 or 100 mg/kg bw/day. Based on the above findings, the NOAEL for maternal toxicity and embryo-foetal development was considered to be 10 mg/kg bw/day.