N-phenyl-N-[(trichloromethyl)thio]benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no indication of toxicity to fertility based on the available data. The NOEL for systemic effects was 500 mg/kg B.W., the highest dose tested, for both sexes in a sub-actute toxicity study. Local irritation of gastric mucosa was observed in the sub-acute toxicity study at 150 mg/kg B.W. leading to a local NOAEL (no-observed-adverse-effect-level) of 50 mg/kg B.W. for male and female rats for N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide after 4-week daily oral treatment by gavage. There is no indication of adverse effects on the reproductive organs in this study. In the developmental toxicity study reported below, there is no indication of maternal or developmental toxicity up to the high dose tested. Overall, there is no trigger to perform a generation study at this tonnage band. No Screening test has to be conducted because a developmental toxicity study according to OECD TG 414 is available.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Quality of whole database:

There is no indication of toxicity to fertility based on the available data. The NOEL for systemic effects was 500 mg/kg B.W., the highest dose tested, for both sexes in a sub-actute toxicity study. Local irritation of gastric mucosa was observed in the sub-acute toxicity study at 150 mg/kg B.W. leading to a local NOAEL (no-observed-adverse-effect-level) of 50 mg/kg B.W. for male and female rats for N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide after 4-week daily oral treatment by gavage. There is no indication of adverse effects on the reproductive organs in this study. In the developmental toxicity study reported below, there is no indication of maternal or developmental toxicity up to the highs dose tested. Overall, there is no indication of adverse effects on fertility based on all available data.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

An OECD TG 414 study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. Based on the results of the study, a dosage of 1000 mg/kg bw/day, the highest dosage tested, was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female and the No Observed Effect Level (NOEL) for the developing conceptus.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 15 March 2017 Experimental Completion Date: 10 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide
Physical State/Appearance: White solid
Date Received: 04 May 2016
Storage Conditions: Room temperature in the dark (used/formulated in light)
Expiry Date: 18 July 2017

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. At the start of treatment (Day 5) the females weighed 197 to 319g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution/suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of Envigo Study Number YJ86PP. These investigations showed that test item formulations were stable for at least twenty one days when stored refrigerated (approximately 4 °C in the dark). Bulk formulations were therefore prepared for the study, divided into daily aliquots and stored at approximately +4 °C in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test item formulation and were analyzed for concentration of N Phenyl N [(trichloromethyl)thio]benzenesulphonamide at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 103-108% of the nominal concentration, confirming accurate formulation.

The test item concetration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

The analytical procedure was previously successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision during studies YJ86PP and HL74FW. The homogeneity and stability of the test item in corn oil was also confirmed in study YJ86PP.

Details on mating procedure:

Time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

The test item was administered from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Frequency of treatment:

Daily

Duration of test:

15 days from day 1 of treatment to day 19.

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Low

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Intermediate

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

24 time mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were chosen based on previous toxicity data, including the N-Phenyl-N-[(trichloromethyl)thio]benzene -sulphonamide: Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo Study number YJ86PP). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Maternal examinations:

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Terminal Investigation
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded

Ovaries and uterine content:

The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included in the calculation of late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus

Fetal examinations:

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal, litter and placental weights and external, visceral and skeletal observations.
Data were first analysed using Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance. Where there was no significance, parametric methodology was applied using one way analysis of variance and, if significant, Dunnett’s multiple comparison test. Where statistical significance was observed, non parametric methodology was applied using Kruskal-Wallis nonparametric analysis of variance; and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test. Due to the non-normal distribution of the data, external, visceral and skeletal observations were generally analyzed using non-parametric methodology.

Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Indices:

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
(number of corporalutea - number of implantations / number of corporalutea) x 100
 
Percentage post-implantation loss was calculated as:
(number of implantations - number of live fetuses / number of implantations) x 100
 
Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = (number of male fetuses / total number of fetuses) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Neither the type, incidence nor distribution of clinical signs observed during the study indicated any systemic effect of treatment at 250, 500 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, occasional incidences of transient increased post-dosing salivation were apparent for the majority of females; this clinical sign was first apparent from Day 13 of gestation. Increased post-dosing salivation is frequently observed when animals are dosed via the oral gavage route and, at the incidence observed, was considered to reflect distaste or slight irritancy of the test item formulations rather than any systemic effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, mean cumulative body weight gain was statistically significantly lower than control from Day 17 of gestation, although there were no statistically significant differences in mean body weight gains apparent for any particular phase of gestation. This lower cumulative body weight gain was supported by lower mean overall body weight gain when adjusted for the weight of the gravid uterus, compared to control, indicating that the observed differences in cumulative body weight gains were not attributable to differences in litter size/weight. Despite the lower cumulative body weight gain, there were no statistically significant differences from control apparent for mean body weight throughout the study.
There was no effect of treatment on body weight or body weight gain during gestation, including when adjustment of final body weight and overall body weight gain was made for the contribution of the gravid uterus, at 250 or 500 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, mean food consumption was statistically significantly lower than control between Days 5 to 8 of gestation. Food consumption was also slightly lower than control from Day 11 of gestation, although differences from control did not attain statistical significance.
There was no effect of treatment on food consumption during gestation at 250 or 500 mg/kg bw/day.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of macroscopic necropsy findings for adult animals at Day 20 of gestation indicated any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for Pre and Post implantantion loss %.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for number of embryonic/fetal deaths.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for early and late numbers of embryonic/ fetal deaths

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for number of late embryonic/ fetal deaths

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable as animals killed on day 20 of gestation
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Not applicable, animals killed on day 20 of gestation

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 1 in section 'Any other information on results' for number of pregnant females.

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effect observed ate the highest dose tested.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no effects of maternal treatment on mean fetal, litter or placental weights at 250, 500 or 1000 mg/kg bw/day.
At 500 mg/kg bw/day, mean placental weight was statistically significantly higher than control but, in the absence of any statistically significant difference at 1000 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to maternal treatment.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See Table 8 in section 'Any other information on results' for mean fetal weights.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for number of live implants

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size and sex ratio at 250, 500 or 1000 mg/kg bw/day.
At 500 mg/kg bw/day, sex ratio (percentage male) was statistically significantly higher than control but, in the absence of any statistically significant difference at 1000 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to maternal treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

See Table 8 in section 'Any other information on results' for mean litter data values.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable as animals killed on day 20 of gestation.

External malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings apparent during external examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings apparent during detailed skeletal examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings apparent during detailed visceral examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Other effects:

not examined

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed at the highest dose tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Table1     Summary of Female Performance

Category	Number of Females at Dose Level (mg/kg bw/day)
	0 (Control)	250	500	1000
Initial Group Size	24	24	24	24
Non-pregnant	2	0	0	1
Pregnant	22	24	24	23
With live young on Day 20 of gestation	22	24	24	23

 

 

Table2     Summary Incidence of Daily Clinical Observations

	Dose Level (mg/kg bw/day)
	0 (Control)	250	500	1000
Number of animals	24	24	24	24
Increased post-dosing salivation	0	0	0	19
Mass on right side of thoracic region	0	1	0	0
Scab on dorsal region	0	0	1	0
No abnormalities detected	24	23	23	5

 

 

Table 3     Group Mean Body Weight Values

Dose Level (mg/kg bw/day)		Body Weight (g) on Day of Gestation
		3	5	6	7	8	11	14	17	20
0 (Control)	mean	249.3	263.1	265.3	271.8	275.5	299.8	316.5	345.0	390.4
	sd	27.6	28.0	28.5	28.0	28.6	30.7	32.0	33.8	39.1
	n	22	22	22	22	22	22	22	22	22
250	mean	251.5	266.0	269.0	274.4	279.5	303.6	319.8	350.0	393.0
	sd	23.6	23.9	23.2	24.2	24.2	26.3	25.7	29.8	32.0
	n	24	24	24	24	24	24	24	24	24
500	mean	250.5	264.8	268.3	273.7	278.8	301.0	317.1	343.0	385.4
	sd	20.9	21.3	21.4	21.7	22.0	23.8	25.3	25.8	31.0
	n	24	24	24	24	24	24	24	24	23
1000	mean	251.3	266.3	269.0	273.7	278.5	300.8	309.6	334.6	378.5
	sd	22.1	22.9	21.9	22.1	22.3	22.6	23.6	21.2	28.8
	n	23	23	23	23	23	23	23	23	23

 

 

Table 4     Group Mean Body Weight Change Values 

Dose Level (mg/kg bw/day)		Body Weight Change (g) during Days of Gestation
		3 to 5	5 to 6	6 to 7	7 to 8	8 to 11	11 to 14	14 to 17	17 to 20
0 (Control)	mean	13.8	2.2	6.5	3.7	24.3	16.8	28.4	45.5
	sd	4.3	2.7	3.5	4.5	5.2	4.9	3.4	6.9
	n	22	22	22	22	22	22	22	22
250	mean	14.5	3.0	5.4	5.1	24.1	16.1	30.3	43.0
	sd	4.0	2.7	2.7	3.2	3.8	5.8	7.4	10.2
	n	24	24	24	24	24	24	24	24
500	mean	14.3	3.5	5.4	5.1	22.3	16.1	25.8	40.7
	sd	4.1	2.5	4.0	2.8	4.7	7.9	8.7	11.4
	n	24	24	24	24	24	24	24	23
1000	mean	15.0	2.7	4.7	4.9	22.3	8.8	25.0	43.9
	sd	3.7	4.3	3.5	3.4	4.4	15.1	11.8	13.0
	n	23	23	23	23	23	23	23	23

Dose Level (mg/kg bw/day)		Cumulative Body Weight Change (g) from Day 5 of Gestation
		5	6	7	8	11	14	17	20
0 (Control)	mean	13.8	2.2	8.7	12.4	36.7	53.5	81.9	127.3
	sd	4.3	2.7	3.0	5.3	6.1	9.2	10.3	14.9
	n	22	22	22	22	22	22	22	22
250	mean	14.5	3.0	8.4	13.5	37.6	53.7	84.0	127.0
	sd	4.0	2.7	2.9	2.9	4.9	7.1	11.0	13.6
	n	24	24	24	24	24	24	24	24
500	mean	14.3	3.5	8.9	14.0	36.3	52.4	78.2	120.1
	sd	4.1	2.5	4.2	4.3	7.6	11.5	15.6	21.0
	n	24	24	24	24	24	24	24	23
1000	mean	15.0	2.7	7.3	12.2	34.5	43.3	68.3**	112.2**
	sd	3.7	4.3	3.6	5.1	5.5	17.2	13.9	18.5
	n	23	23	23	23	23	23	23	23

 

Table 5     Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)		Body Weight (g) on Days of Gestation		Body Weight Change (g) during Days of Gestation	Gravid Uterus Weight (g)	Adjusted Body Weight (g)  Day 20	Adjusted Body Weight Change (g) 5-20
		5	20	5-20
0 (Control)	mean	263.9	392.0	128.1	83.765	308.2	44.3
	sd	28.5	39.3	14.8	12.017	32.7	10.3
	n	21	21	21	21	21	21
250	mean	265.0	391.6	126.7	84.550	307.1	42.1
	sd	23.8	31.9	13.8	15.509	27.3	12.4
	n	23	23	23	23	23	23
500	mean	265.3	385.4	120.1	79.373	306.1	40.7
	sd	21.6	31.0	21.0	18.763	24.8	11.8
	n	23	23	23	23	23	23
1000	mean	265.5	376.6	111.1**	85.787	290.8	25.3***
	sd	23.2	28.1	18.2	11.368	24.5	17.0
	n	22	22	22	22	22	22

 

 

Table 6     Group Mean Food Consumption Values

Dose Level (mg/kg bw/day)		Food Consumption (g/rat/day) between Days of Gestation
		3 - 5	5 - 8	8 - 11	11 - 14	14 - 17	17 - 20
0 (Control)	mean	23.5	21.1	22.4	23.4	25.7	26.0
	sd	3.1	2.1	2.6	2.6	2.9	3.2
	n	22	22	22	22	22	22
250	mean	24.5	21.1	22.5	24.3	25.9	26.6
	sd	2.3	2.1	3.0	4.5	3.0	3.3
	n	24	24	24	24	24	24
500	mean	24.5	20.3	22.7	22.8	23.6	27.5
	sd	2.3	1.7	2.3	3.4	4.4	4.6
	n	24	24	24	24	24	24
1000	mean	23.3	19.5**	23.1	21.1	23.6	24.5
	sd	6.0	1.7	2.2	5.0	3.3	3.8
	n	23	23	23	23	23	23

 

 

Table 7     Summary Incidence of Necropsy Findings

	Dose Level (mg/kg bw/day)
	0 (Control)	250	500	1000
Number of animals examined	24	24	24	24
Mammary tissue:Solid mass attached, located under right forelimb	0	1	0	0
Stomach:Gaseous distension	0	1	0	0
No abnormalities detected	24	22	24	24

 

Table 8     Group Mean Litter DataValues

Dose Level (mg/kg bw/day)		Number of Corpora Lutea	Number of Implants	Number of Embryonic/Fetal Deaths			Implantation Loss %		Number of Live Implants			% MaleFetuses	Mean Male Fetal Weight (g)	Mean Female Fetal Weight (g)	Mean Fetal Weight (g)	MeanPlacentalWeight (g)	Litter Weight (g)	TotalPlacentalWeight (g)
				Early	Late	Total	Pre	Post	Male	Female	Total
0 (Control)	mean	16.5	13.9	0.1	0.1	0.3	15.6	2.8	6.1	7.3	13.5	45.2	4.072	3.886	3.971	0.557	53.415	7.475
	sd	3.0	2.1	0.4	0.4	0.5	13.4	4.6	1.9	1.5	2.1	10.1	0.218	0.195	0.197	0.048	8.520	1.170
	n	22	22	22	22	22	21	22	22	22	22	22	22	22	22	22	22	22
250	mean	16.4	14.0	0.1	0.1	0.2	14.2	3.1	6.6	6.9	13.5	49.0	4.141	3.890	4.015	0.566	54.040	7.620
	sd	2.7	2.7	0.3	0.4	0.5	14.9	6.2	2.4	2.3	2.8	13.5	0.405	0.349	0.357	0.062	10.806	1.676
	n	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24
500	mean	16.1	12.8	0.1	0.1	0.2	20.0	1.8	7.0	5.6	12.6	55.4*	4.250	3.980	4.123	0.610*	51.786	7.499
	sd	3.0	3.3	0.3	0.3	0.4	20.1	4.1	2.4	2.5	3.3	14.3	0.311	0.340	0.320	0.087	13.580	1.817
	n	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24
1000	mean	16.7	14.3	0.1	0.0	0.1	13.1	0.9	7.6	6.5	14.1	54.3	4.020	3.775	3.907	0.593	55.136	8.333
	sd	2.9	2.0	0.3	0.0	0.3	11.7	2.5	1.9	2.2	2.0	13.0	0.321	0.293	0.290	0.066	8.360	1.228
	n	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23

 

 

Table 9     Summary Incidence of Fetal External Findings

External Findings	Dose level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of fetuses (litters) examined
	296 (22)			325 (24)			303 (24)			325 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
Total Number Affected	3	3	1.2	1	1	0.3	10	6	6.4	6	4	1.9
Small	2	2	0.8	1	1	0.3	4	2	1.3	6	4	1.9
Pale	1	1	0.3	0	0	0.0	1	1	0.3	0	0	0.0
Swollen abdomen	0	0	0.0	0	0	0.0	1	1	0.3	0	0	0.0
Placenta small	0	0	0.0	1	1	0.3	1	1	0.3	1	1	0.3
Placenta large	1	1	0.4	0	0	0.0	4	2	4.5	0	0	0.0

 

Table 10   Summary Incidence of Fetal Visceral Findings

Visceral Findings	Dose Level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of Fetuses (litters) Examined
	155 (22)			169 (24)			159 (24)			169 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
HEAD
Rugae - non-uniform patterning	13	11	9.1	10	9	5.6	10	8	8.2	7	5	4.8
ABDOMEN
Liver - additional lobe between right and left median	0	0	0.0	1	1	0.5	1	1	0.6	1	1	0.5
Spleen - reduced in size	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.5
Umbilical artery - left-sided	1	1	0.5	1	1	0.6	0	0	0.0	1	1	0.5
Urinary bladder - distended with fluid or gas	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.6
Testis - partially undescended	0	0	0.0	1	1	1.0	1	1	0.7	1	1	0.9
Ureter - kinked	15	10	9.8	6	6	3.2	8	7	6.1	10	8	5.7
Ureter - dilated - Slight/Severe	11	7	6.5	4	4	2.2	6	6	3.3	7	6	4.1
Renal pelvic cavitation - increased - Slight/Severe	6	4	4.1	0	0	0.0	5	4	4.5	1	1	0.6
Renal papilla - absent	2	2	1.3	0	0	0.0	0	0	0.0	0	0	0.0
THORAX
Thymus - lobe partially undescended	2	2	1.3	6	6	3.4	5	5	4.2	3	3	1.6
Innominate artery - absent	0	0	0.0	1	1	0.6	0	0	0.0	0	0	0.0
Atrium - enlarged	0	0	0.0	0	0	0.0	1	1	0.6	0	0	0.0
Total	31	19	20.6	25	15	13.9	22	15	16.4	23	14	13.8

 

Table 11   Summary Incidence of Fetal Skeletal Findings

Skeletal Findings	Dose Level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of Fetuses (litters) Examined
	141 (22)			156 (24)			144 (24)			156 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
SKULL
Fontanelle (anterior) - large	1	1	0.8	0	0	0.0	2	2	1.4	3	3	2.0
Nasal - incomplete ossification	8	6	6.4	14	9	8.0	3	2	1.9	6	5	3.9
Frontal - incomplete ossification	0	0	0.0	2	2	1.1	2	2	1.3	1	1	0.6
Frontal - unossified area	1	1	0.6	2	1	1.2	0	0	0.0	3	3	1.8
Frontal - sutural line	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.5
Parietal - incomplete ossification	6	5	4.8	5	5	3.1	4	4	2.6	4	3	2.5
Parietal/Interparietal - sutural bone	1	1	0.6	0	0	0.0	0	0	0.0	1	1	0.6
Interparietal - incomplete ossification	15	8	11.8	14	10	8.3	6	3	4.2	12	10	7.9
Interparietal - unossified area(s)	1	1	0.8	1	1	0.5	0	0	0.0	0	0	0.0
Occipital (Supra-occipital) - incomplete ossification	3	3	2.7	9	9	5.2	2	2	1.4	8	6	5.1
Occipital (Supra-occipital) - unossified area(s)	2	2	1.6	3	3	1.6	6	5	5.6	4	4	2.5
Squamosal - incomplete ossification	9	6	6.0	8	7	4.6	6	6	3.9	7	6	4.3
Squamosal - unossified area(s)	1	1	0.6	5	3	2.7	5	4	2.9	0	0	0.0
Jugal - incomplete ossification	4	3	3.3	3	3	1.7	2	2	1.4	1	1	0.6
Zygomatic process of maxilla - incomplete ossification	7	5	4.8	5	5	3.2	3	3	2.0	3	3	1.8
Zygomatic process of squamosal - incomplete ossification	1	1	0.9	1	1	0.6	1	1	0.7	1	1	0.6
Premaxilla - incomplete ossification	1	1	0.9	0	0	0.0	0	0	0.0	1	1	0.6

 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings	Dose Level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of Fetuses (litters) Examined
	141 (22)			156 (24)			144 (24)			156 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
SKULL
Hyoid - incomplete ossification	8	5	6.0	8	6	4.6	6	5	4.0	4	4	2.5
Hyoid - not ossified	4	4	2.4	6	4	3.3	4	4	2.7	10	4	6.3
Presphenoid - incomplete ossification	0	0	0.0	0	0	0.0	1	1	0.7	0	0	0.0
Presphenoid - not ossified	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.6
Basisphenoid - incomplete ossification	1	1	0.9	2	2	1.6	0	0	0.0	0	0	0.0
VERTEBRAL COLUMN
Odontoid - ossification present	0	0	0.0	1	1	0.6	2	1	1.4	0	0	0.0
Ventral arch of vertebra 1 - ossification present	52	19	36.1	59	19	36.0	64	19	47.5	64	21	40.8
Cervical (neural) arch - incomplete ossification	1	1	0.9	2	2	1.1	6	6	3.8	9	5	5.7
Thoracic centrum - incomplete ossification	1	1	0.8	8	5	4.5	11	6	8.3	4	3	2.6
Thoracic centrum - not ossified	0	0	0.0	2	1	1.0	0	0	0.0	0	0	0.0
Thoracic centrum - bipartite ossification	2	2	1.5	2	2	1.2	5	4	3.3	2	2	1.3
Thoracic centrum - dumb-bell-shaped	11	8	7.2	12	10	7.0	21	11	14.7	7	5	4.4
Thoracic centrum - asymmetrically ossified	1	1	0.6	2	2	1.3	4	3	2.6	1	1	0.7
Lumbar centrum - dumb-bell-shaped	0	0	0.0	0	0	0.0	1	1	0.7	0	0	0.0
Lumbar centrum - asymmetrically ossified	0	0	0.0	0	0	0.0	1	1	0.7	0	0	0.0
Sacral (neural) arch - incomplete ossification	4	4	3.0	8	5	4.8	7	5	4.6	13	7	8.6
Sacral (neural) arch - not ossified	0	0	0.0	1	1	0.5	0	0	0.0	0	0	0.0
Caudal vertebrae - less than 4 ossified	12	5	8.5	18	8	10.1	14	8	8.7	14	7	9.2

 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings	Dose Level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of Fetuses (litters) Examined
	141 (22)			156 (24)			144 (24)			156 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
RIBS
Ossification centre - associated with 7th cervical vertebra	0	0	0.0	1	1	0.5	1	1	0.7	3	3	1.9
Ossification centre - associated with 1st lumbar vertebra	3	2	1.8	6	5	4.0	12	7	7.8	11	6	7.1
One or more ribs - thickened	1	1	0.9	0	0	0.0	0	0	0.0	1	1	0.6
Rib - short	0	0	0.0	0	0	0.0	1	1	0.6	0	0	0.0
Rib - rudimentary	1	1	0.9	2	2	1.3	0	0	0.0	1	1	0.5
Rib - misshapen	0	0	0.0	0	0	0.0	1	1	0.5	0	0	0.0
Costal cartilage - misaligned	1	1	0.6	3	3	1.6	3	3	2.0	8	6	4.8
Costal cartilage - not fused to sternebra	12	7	9.6	14	10	9.4	9	6	5.7	7	5	4.2
STERNEBRAE
Sternebra - incomplete ossification	0	0	0.0	2	2	1.0	1	1	0.6	1	1	0.6
Sternebra - not ossified	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.6
Sternebra - bipartite ossification	1	1	0.9	0	0	0.0	0	0	0.0	2	2	1.2
Sternebra - misaligned	5	4	3.4	5	4	3.3	5	5	3.3	5	4	3.3
Sternum - split	0	0	0.0	1	1	1.0	1	1	0.5	1	1	0.6
Sternum - split - cartilage only	0	0	0.0	1	1	1.0	0	0	0.0	0	0	0.0
Xiphoid cartilage - split	0	0	0.0	1	1	1.0	1	1	0.5	0	0	0.0
Xiphoid cartilage - partially split	4	4	2.9	14	11	9.4	9	5	5.8	7	6	4.6

 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings	Dose Level (mg/kg bw/day)
	0 (Control)			250			500			1000
	Number of Fetuses (litters) Examined
	141 (22)			156 (24)			144 (24)			156 (23)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
PECTORAL GIRDLE
Scapula - misshapen	8	5	5.7	2	2	1.3	6	5	3.8	5	3	3.2
PELVIC GIRDLE
Ischium - incomplete ossification	0	0	0.0	0	0	0.0	0	0	0.0	1	1	0.6
Pubis - not ossified	0	0	0.0	1	1	0.5	0	0	0.0	0	0	0.0
Pubis - incomplete ossification	2	1	1.5	1	1	0.6	2	2	1.3	6	3	3.7
FORELIMBS
Metacarpal - not ossified	16	11	11.7	17	11	10.2	21	10	13.3	27	11	17.2
Metacarpal - incomplete ossification	0	0	0.0	0	0	0.0	0	0	0.0	2	2	1.2
Forepaw phalanges - 1 or more - ossified	26	11	18.6	46	17	30.5	41	18	31.7	53	14	32.1
Humerus - incomplete ossification	3	3	2.0	0	0	0.0	1	1	0.7	1	1	0.6
Humerus - hole	2	2	1.4	0	0	0.0	2	2	2.0	0	0	0.0
HINDLIMBS
Metatarsal - 1st - ossified	4	3	2.8	3	3	1.8	4	3	2.7	6	3	3.5
Metatarsal - incomplete ossification	0	0	0.0	0	0	0.0	2	2	1.4	0	0	0.0
Femur - incomplete ossification	7	4	5.2	5	4	3.0	6	5	6.8	11	4	6.8
Tarsal flexure - cartilage – severe	0	0	0.0	1	1	0.6	0	0	0.0	0	0	0.0
Total	111	22	79.1	134	24	84.2	120	23	85.3	129	23	82.0

NOTE: a fetus may appear in more than one category

Conclusions:

Based on the results of the study, a dosage of 1000 mg/kg bw/day, the highest dosage tested, was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female and the No Observed Effect Level (NOEL) for the developing conceptus.

Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 250, 500 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Clinical signs were generally restricted to occasional incidences of transient increased post-dosing salivation for the majority of females at 1000 mg/kg bw/day and did not indicate any systemic effect of treatment.  

Body Weight

At 1000 mg/kg bw/day, mean cumulative body weight gain was statistically significantly lower than control from Day 17 of gestation and overall body weight gain remained statistically significantly lower than control, when adjusted for the contribution of the gravid uterus. 

There was no effect of treatment on body weight or body weight gain during gestation, including when adjustment of final body weight and overall body weight gain was made for the contribution of the gravid uterus, at 250 or 500 mg/kg bw/day.

Food Consumption

At 1000 mg/kg bw/day, mean food consumption was statistically significantly lower than control between Days 5 to 8 of gestation. 

There was no effect of treatment on food consumption during gestation at 250 or 500 mg/kg bw/day.

Post Mortem Studies

Macroscopic necropsy findings for adult animals at Day 20 of gestation did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Litter Responses

Litter Data

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size and sex ratio at 250, 500 or 1000 mg/kg bw/day.

Litter, Placental and Fetal Weights

There were no effects of maternal treatment on mean fetal, litter or placental weights at 250, 500 or 1000 mg/kg bw/day.

Fetal Evaluation

External Examinations

External examination of fetuses at Day 20 of gestation did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Detailed Visceral Examinations

Detailed visceral examinations of fetuses at Day 20 of gestation did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Detailed Skeletal Examinations

Detailed skeletal examinations of fetuses at Day 20 of gestation did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Conclusion

Based on the results of the study, a dosage of 1000 mg/kg bw/day, the highest dosage tested, was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female and the No Observed Effect Level (NOEL) for the developing conceptus.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

An OECD TG 414 study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD TG 414 study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 250, 500 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Conclusion

Based on the results of the study, a dosage of 1000 mg/kg bw/day, the highest dosage tested, was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female and the No Observed Effect Level (NOEL) for the developing conceptus.

Justification for classification or non-classification

No classification is warranted; based on the results of the study, a dosage of 1000 mg/kg bw/day, the highest dosage tested, was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female and the No Observed Effect Level (NOEL) for the developing conceptus.