4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening for reproductive / developmental toxicity

The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2008 to 13 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guideline in compliance with GLP and reported with a valid GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals: 48 adult males and 48 females of Wistar rats from Velaz Praha, Czech Republic were used. The animals were acclimatised for 20 days before the study in the condition identical to the condition during the experiment. The acclimatisation was made according to SOPB-00188-AH, Animals quarantine. The mean bodyweight of animals at the start of experiment was in males 269.8g, in females 185g. Twelve males and twelve females were used in the control and in all dose groups. Husbandry: All animals were kept in cages with bedding in the experimental animal house with standard conditions. The sanitation was made according to standard operation procedure. The temperature was 22± 2°C, relative humidity 45 to 65%. They were followed by a room thermometer and hydrometer placed in the room. The room is equipped with central air-conditioner. The light regime was artificial 12-hour light / 12-hour dark cycle. The keeping of animals was organised to timetable:1. 4 animals in the cage (males and female separately)2. 1 male and 1 female in the cage (mating)3. 1 pregnant female individually4. 1 female and offspring individuallyFood and water: For feeding conventional laboratory diet (Top Dovo, PD Horné Dubové – Naháč) was used with an unlimited supply of drinking water.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Control article/VehicleIdentification: Olive oil (Olivae oleum raffinatum), product by G. HeessLot number: L 809182Purity: Quality of control article was declared with Certificate.Storage condition: The control article was stored under laboratory conditions in dark container. Justification for choice of vehicle: Dusantox 86 is not soluble in water, but olive oil was used as vehicle. Method of application: The test article was administered per os with the metal stomachic tube everyday at 8 a.m. Doses were calculated on the current bodyweight. The animals were weighed weekly. The fixed application volume of 0.5mL/100g bodyweight was used. Dusantox 86 is not soluble in water; the olive oil was used as a vehicle. The test substance was soluble in olive oil as vehicle every day. The vehicle was applied to control animals in volume 0.5mL/100g bodyweight.

Details on mating procedure:

Normally, 1:1 (1 male to 1 female) mating was used in this study. The female was place with the same male until pregnancy occurs or 14 days have elapsed. Every morning during the mating period the females were examined for the presence if sperm or vaginal plugs. Day 0 of pregnancy was defined as the day a vaginal plug or sperm are found. The pregnant female was kept individually up to 4th day post-partum.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not specified.

Duration of treatment / exposure:

Males - 28 daysFemales - up to four days after birth (54 days)

Frequency of treatment:

Daily

Details on study schedule:

Daily dosing of both sexes began 14 days before mating. Dosing was continued in both sexes during mating period (max. 14 days – 2 complete oestrus cycles). Males were dosed after the mating period at least until the minimum total dosing period of 28 days. They were sacrificed by the overdose of anaesthetic (dietylether). Daily dosing of females were continued throughout pregnancy and up to the 4th day after birth. Female showing no evidence of copulation were dosed 29 days after the last day of mating period. They were sacrificed by overdose of anaesthetic. Females with offpring were sacrificed on day 4 post-partum.

Remarks:

Doses / Concentrations:0, 5, 25 & 50 mg/kg bw/dayBasis:actual ingested

No. of animals per sex per dose:

12 animals per sex, per dose. (Total of 48 rats)

Control animals:

yes, concurrent vehicle

Details on study design:

Groups Designation and Dose Levels: Three test groups and control group (vehicle) were used. Twelve animals of both sexes were used in every level group and in the control group.The animals were marked by serial number 1 – 48. All cages were marked with the number of the group, the dose and the serial number of the animals. The animals in the cage were marked by line I – IIII on the tail. Males were coloured with black and females with red. Selection of doses was estimated from available information about the test article. The low dose was estimated for 5mg.kg-1. The high dose level – 50mg.kg-1 represents 10-fold of the low dose level, medium dose level – 25mg.kg-1 represents 5-fold of the low dose level. Males (black)Females (red)ControlOlive oil(12) 1 -12(12) 1 – 12Low dose group5mg.kg-1(12) 13 – 24(12) 13 – 24Medium dose group25mg.kg-1(12) 25 – 36(12) 25 – 36High dose group50mg.kg-1(12) 37 – 48(12) 37 – 48

Positive control:

Not utilised in this study.

Parental animals: Observations and examinations:

General clinical observations were made once a day 1 hour after the test application. The health condition of the animals, reaction of the animals to the applied substance, their condition were monitored every day and recorded. The number of pregnant females was recorded.Bodyweight and Food Consumption: Individual weighing of the adult animals was performed once a week. The bodyweights of animals were recorded.

Oestrous cyclicity (parental animals):

Not measured

Sperm parameters (parental animals):

Not measured

Litter observations:

The number of live pups, weight of litters and sex of pups were observed.

Postmortem examinations (parental animals):

The animals were anesthetized with diethylether and necropsied. Males: at the end of the mating period (after 28 days of treatment period) and pregnant females after 4 days post-partum and non-pregnant females 28 days after mating period. Kidneys, testes, epididymides, uterus and ovaries were weighed during the necropsy. The samples from these organs with prostate and glands vesicles were processed for microscopic examination. Those tissue samples were fixed in bouin solution and prepared in the paraffin technical. The tissue segments were sectioned at 10μm by a Leica micrptome and stained with hematoxylin and eosin. The histological section was examined under a light microscope. Macroscopic Evaluation: At the time of death the animals were examined macroscopically for any abnormalities or pathological changes. Special attention was given to the organs of the reproductive system and the kidneys as target organ. The number of corpora lutea and count of implantation sites was monitored. Histopathological Examination: Histopathological evaluations have a prominent role in reproductive toxicity assessment. Detailed histological examination was performed on the uterus, ovaries, testes, epididydimes and kidneys (the target organ) of the animals of control and highest (50mg.kg-1) dose groups. The histopathological examination was made by standard operation procedures.

Postmortem examinations (offspring):

Postmorten exmainations not performed on offspring.

Statistics:

The results obtained during the study were statistically evaluated by statistical programme Statgraphics. Statistical evaluated was made separately for males, females and litters. For identification homogeneity groups were used the Bartllet’s test. In the case of homogeneity One-Way Analysis of variance with consecutive Multiple Rangers tests was accomplished. In case non homogeneity Kruskal-Wallis One Way Analysis by Ranks was applied.

Reproductive indices:

After sacrificed of all females were calculated reproduction indices Number of females with implantsFertility Index = -------------------------------- x 100 Number of females mating Number of females delivering live youngGestation (Pregnancy) Index = --------------------------------------------- x 100 Number of females with evidence of pregnancy Number of live offspringLive Birth Index = --------------------------------- x 100 Number of offspring delivered

Offspring viability indices:

Number of live offspring at lactation day 4Viability (Survival) Index = --------------------------------------------- x 100Number of live offspring delivered

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mating Procedure: Twelve animals both sexes were used in every level groups. Every female were placed with a single male from the same dose level (1:1 mating) until copulation (1 to 14 days). Females were examined for presence of sperm or vaginal plug daily at 6:30 a.m. The day 0 of the pregnancy was defined as the day presence of vaginal plug or sperm. The copulation of the majority of females in control and all dose groups were established in the first five days. Pregnancy: Twelve pairs in every dose group and in the control group were started. The evidence of copulation was demonstrated in control and in dose group 50mg.kg-1 in twelve females. The sperm of vaginal plugs were found in nine females in dose group 5mg.kg-1 and in eleven females in dose group 25 mg.kg-1. In three females (No 14, 15 & 24) in dose group 5mg.kg-1 and one female (No 30) in dose group of 25 mg.kg-1 were not observed the sperm or vaginal plugs. These females were not pregnant. The majority of females length of pregnancy were 23 days – seven female in the control and the dose group 50mg.kg-1, four animals in the dose group 5mg.kg-1 and six in the dose group of 25mg.kg-1. The length of pregnancy 22 days was established in three females in the control group, in five females in the dose group 5 and 25mg.kg-1 and in four females in the dose group 50mg.kg-1. 21 days of pregnancy were established in one female in the control and the dose group of 50 mg.kg-1. Pathology: Histopathological evaluation have a prominent role in reproductive toxicity assessment. After finishing the test all animals were sacrificed by overdose of anaesthetic and moved to histological evaluation. Special attention was paid to the reproductive system and to the kidneys as target organ. Pathology Results and DiscussionStatistical Analysis of Organ weight Data: Relative weights of organs in the treated animals were not significantly different from those in the control group.Relative weight of observed organs was calculated from formula: A (g)R (%) = ------------ x 100 Weight (g) Gross Pathology (Macroscopical Evaluation): All animals survived until the necropsy. One macerate dead foetus (prenatal age about 18-19 days) was found in the female No 9 from control group in cornis left of uterus. An individual cyst of 2mm was observed in the ovary left of female No 39 from 50mg.kg-1 dose group. Strinking structura of liver tubuli in female No 45 and a change of liver colouration (light brown) in female No 48 were observed, both females from 50mg.kg-1 dose group. Histopathological ExaminationLiver: Several small mononuclear nodules in parenchyma of liver were observed in female No 45 from 50mg.kg-1 dose group. Moderate diffuse small-droplet vacuolization of hepatocytes and individual mononuclear nodules were found in female No 48 950 mg.kg-1 dose group). Kidneys: Focal occurrence of caryopycnosis was seen in 3 females (No 37, 43 & 46) of 50 mg.kg-1 dose group and in 1 female (No 6) from control group. Small focal interstitial lymphocytic infiltrations were observed in 1 (No 4) female and in 1 male (No 5) from the control group. Sporadic glomerular atrophy was found in 1 female (No 44) and in 3 males (No 38, 41 & 44) of 50 mg.kg-1 dose group. Uterus: Focal accumulation of macrophages with yellowish pigment was observed in 1 female (No 1) in the control group. Inflammatory cells infiltration of mucosa and focal inflammatory lesions in submucosa were seen in 1 female (No 9) in cornis left. This lesion was associated with finding of dead foetus. Testes: Hypoplasia of existing cells in the several seinoferous tubles and marked loss of spermatogenic cells were observed in male No 9 in the control group. Epididymis: Moderate perivascular interstitial lymphocytic infiltration was seen in male No 45 in the 50 mg.kg-1 dose group. Prostate: Focal small interstitial lymphocytic lesions were found in 1 male of the control group (No 3) and in 3 males in the 50 mg.kg-1 dose group (No 41, 46 & 48).In the remaining organs and tissues no histopathologic changes were observed. Discussion of Pathological Results: The pathological examination has shown no extensive changes in organs and tissues of rats. The nature and frequency of lesions in the animals in the high dose groups were similar to the control group. We have not seen microscopical change related to the effect of Dusantox 86.

Dose descriptor:

NOEC

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Alive and dead pups were counted on days 1 and 4. In the control group in female No 3 after birth two pups dead and in female No 9 one death macerate foetus was established. 102 alive and seven dead pups were born in this group. In the dose group 5mg.kg-1 in female No 18 after birth two dead pups were observed. 79 alive and 4 dead pups were in this group. Three pups dead were seen in female No 32 after birth on the dose group 25mg.kg-1. In this group 79 alive and 11 dead pups were born. In the dose group 50mg.kg-1 112 alive and one dead pups were born. In the control and also dose group of 25mg.kg-1 and 50mg.kg-1 were observed two dead pups after 4th day. There was observed in the control group in one female mean count of live pups 9 and in dose group 5 mg.kg-1 and 50 mg.kg-1 and 7 in the dose group 25mg.kg-1 after 1st day. There was seen at one female count of live pups 8 in the control group, 9 in dose group 5mg.kg-1 and 50 mg.kg-1 and 7 in the dose group 25 mg.kg-1 after 4th day. The litters were weighed on day 1 and 4. Mean weight of litter after birth was 53.33g in the control group, 51.67g in the dose group 5mg.kg-1, 43.64g in the dose group 25mg.kg-1 and 58.75 in the dose group 50mg.kg-1. Mean weight of litter at 4th day was 70.83g in the control group, 75.56g in the dose group 5mg.kg-1, 61.356g in the dose group 25mg.kg-1 and 77.92g in the dose group 50mg.kg-1. Offspring were sexed on the 4th day post-partum. Mean sex ratio was 4 males/4 females in the control and in the dose group 5mg.kg-1, 3 males/4females in the dose group 25mg.kg-1 and 4 males/5 females in the dose group 50mg.kg-1.

Dose descriptor:

NOEC

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

Reproductive effects observed:

not specified

Table 1. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Males

Dose level	N	Bodyweight (g) / Day
		1	7	14	21	28
Control	12/12	272.50± 12.88	286.17± 15.05	301.67± 20.38	321.67± 21.67	330.00± 26.63
Low 5mg.kg^-1	12/12	265.83± 16.21	280.83± 16.21	295.00± 15.08	319.17± 18.32	325.83± 18.81
Medium 25mg.kg^-1	12/12	270.00± 19.54	287.50± 23.79	299.17± 25.75	326.67± 29.65	331.67± 30.40
High 50mg.kg^-1	12/12	270.83± 15.64	281.67± 19.92	299.17± 27.12	322.50± 31.66	329.17± 31.18

 

Table 2. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pre-Mating Period, Females

Dose level	N	Bodyweight (g) / Day
		1	7	14
Control	12/12	185.83± 9.96	186.67± 10.73	190.83± 9.00
Low 5mg.kg^-1	12/12	186.67± 9.85	185.83± 10.84	190.00± 11.28
Medium 25mg.kg^-1	12/12	183.33± 11.55	185.00± 7.98	188.33± 10.30
High 50mg.kg^-1	12/12	184.17± 7.93	186.67± 12.31	188.33± 10.30

 

Table 3. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Mating Period, Females

Dose level	N	Bodyweight (g) / Day
		1	7	14
Control	12/12	198.33± 9.37	215.83± 12.40	235.00± 11.68
Low 5mg.kg^-1	12/12	199.17± 10.84	216.67± 16.14	232.50± 23.79
Medium 25mg.kg^-1	12/12	198.33± 11.93	209.17± 13.11	230.83± 14.43
High 50mg.kg^-1	12/12	200.83± 11.65	210.83± 12.40	230.83± 11.65

 

Table 4. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pregnancy, Females

Dose level	N	Bodyweight (g) / Day
		1	14	21
Control	12/12	200.00± 9.54	235.00± 11.68	281.67± 27.1
Low 5mg.kg^-1	12/12	202.22± 9.72	240.00± 18.71	288.89± 28.92
Medium 25mg.kg^-1	12/12	208.18± 13.28	232.73± 14.89	280.00± 24.08
High 50mg.kg^-1	12/12	210.83± 12.40	232.50± 14.22	283.33± 25.70

 

N-Number of animals, the results are presented as mean± SD

 

Table 5. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, F1 generation (Offspring)

Dose level	Bodyweight (g) / Day
	N	1	N	4
Control	102	53.33± 30.25	101	70.83± 38.49
Low 5mg.kg^-1	78	51.67± 23.98	78	75.56± 34.68
Medium 25mg.kg^-1	79	43.64± 23.99	77	61.36± 35.08
High 50mg.kg^-1	112	58.75± 17.34	110	77.92± 25.89

N-Number of pups, the results are presented as mean± SD

 

Table 6. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Mean Number of Corpora Lutea and Implantation Sites

Dose level	N	Corpora Lutea	Implantation Sites
Control	12/12	13.00± 2.13	11.08± 3.50
Low 5mg.kg^-1	12/9	10.67± 1.41	10.33± 1.00
Medium 25mg.kg^-1	12/11	11.73± 1.56	11.36± 1.21
High 50mg.kg^-1	12/12	12.58± 2.39	11.83± 1.95

N-Number of females, the results are presented as mean± SD

 

Table 11. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Males

Dose level	N	Kidneys (g)		Testis (g)		Epididymis (g)
		Right	Left	Right	Left	Right	Left
Control	12/12	0.36± 0.03	0.36± 0.02	0.46± 0.05	0.48± 0.07	0.089± 0.013	0.090± 0.010
Low 5mg.kg^-1	12/12	0.36± 0.03	0.35± 0.03	0.48± 0.04	0.47± 0.04	0.086± 0.011	0.089± 0.012
Medium 25mg.kg^-1	12/12	0.36± 0.03	0.35± 0.03	0.46± 0.06	0.47± 0.06	0.088± 0.011	0.085± 0.010
High 50mg.kg^-1	12/12	0.38± 0.04	0.37± 0.04	0.49± 0.05	0.51± 0.07	0.094± 0.011	0.097± 0.011

 

Table 12. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Females

Dose level	N	Kidneys (g)		Ovaries (g)		Uterus (g)
		Right	Left	Right	Left
Control	12/12	0.037± 0.03	0.37± 0.02	0.38± 0.06	0.36± 0.07	0.29± 0.09
Low 5mg.kg^-1	12/12	0.37± 0.04	0.35± 0.04	0.40± 0.08	0.41± 0.10	0.27± 0.04
Medium 25mg.kg^-1	12/12	0.37± 0.03	0.36± 0.03	0.38± 0.11	0.38± 0.09	0.29± 0.07
High 50mg.kg^-1	12/12	0.38± 0.04	0.36± 0.04	0.34± 0.03	0.37± 0.05	0.27± 0.05

 

N-Number of animals, the results are presented as mean± SD

 

Table 13. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Percentage Evaluation of Results in Females

Dose level	N	Sperm-positive (%)	Pregnant (%)	With Live Pups (%)
Control	12/12	100	100	83.33
Low 5 mg.kg^-1	12/9	75	75	66.67
Medium 25mg.kg^-1	12/11	91.67	91.67	83.33
High 50mg.kg^-1	12/12	100	100	100

N-Number of females

 

Table 14. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – F1 Generation (Offspring)

Dose level	Number of Pups				Live Pups (%)	Death Pups (%)
	Total	Mean/1 Female	Live	Death
Control	109	9	102	7	93.58	6.42
Low 5mg.kg^-1	82	9	78	4	95.12	4.88
Medium 25mg.kg^-1	90	8	79	11	87.78	12.22
High 50mg.kg^-1	113	9	112	1	99.12	0.89

 

Table 15. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Indices of Reproductive Parameters (%)

Dose level	Fertility	Gestation	Live Birth	Viability
Control	100	83.33	93.58	98.80
Low 5mg.kg^-1	75	88.89	95.12	100
Medium 25mg.kg^-1	91.67	90.91	87.78	97.47
High 50mg.kg^-1	100	100	99.12	98.21

 

Table 16. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Sex Distribution of Pups

Dose level	Total Number	Males	%	Females	%
Control	100	53	53.00	47	47.00
Low 5mg.kg^-1	78	37	47.44	41	52.56
Medium 25mg.kg^-1	77	36	46.75	41	53.25
High 50 mg.kg^-1	110	49	49.00	61	61.00

Sex distribution – 4^thday post-partum

Conclusions:

The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

Executive summary:

This study was used to provide initial information on possible effects on reproduction and/or development, which may be evoked by Dusantox 86. The Wistar rats, 48 adult males and 48 adult females were divided into four groups. Three groups received the test article Dusantox 86 in graduated doses. The control group received the vehicle. Dusantox 86 is not soluble in water; olive oil was used as a vehicle. The dose of 5mg.kg^-1was estimated as the low dose, the dose of 25mg.kg^-1as the medium dose level and as the high dose level was selected 50mg.kg^-1. Doses were calculated on the current bodyweight. The test article and vehicle were administered per os by metal stomachic tube every day. The males were applied 28 days – 14 days per-mating, 14 days mating. This dosing period (28 days) is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis. The females were applicated 54 days – 14 days pre-mating, 14 days mating (with the objective of covering at least two complete oestrus cycles), 22 days gestation, and 4 days lactation. The application volume 0.5mL/100g weight was administered. All animals were monitored for signs of toxicity during application of the test article. All animals were weighed weekly. The bodyweight, food consumption and clinical observation were observed during the study. After finishing the test all animals were sacrificed by overdosing of anaesthetic and moved to macroscopical and histological evaluation. The organs of the reproductive system (the uterus, ovaries, testes, epididydimes, and accessory sex organs) and the kidneys as target organ were monitored. The number of implantation sites and corpora lutea was recorded. The results were elaborated in for of tables. The statistical programme Statgraphics was used for statistically evaluation of the results.

There were no test article-related deaths of animal during the study. There were not any visible signs of intoxication in all animals during clinical observation. The incidence of smooth stool in one animal of dose group 50mg.kg^-1males and diarrhoea in one male of dose group 25mg.kg^-1were observed. The incidence of alopecia in one female dose group of 50mg.kg^-1was observed. These incidences were temporary and may not be indicate relation to the test application. The bodyweight of all animals moderately increased during the study. There were no significant differences between all dose groups and the control groups.

The food consumption was adequate. In control (olive oil) and highest (50mg.kg^-1) dose groups 12 females were pregnant. In dose group 5mg.kg^-19 females were pregnant and in dose group 25mg.kg^-111 females were pregnant. In female dose group 5mg.kg^-1three animals and in dose group 25 mg.kg^-1one animal were not pregnant. The number of alive pups in all females of the control and all dose groups was stable. The relative weight of kidneys and reproduction organs in both sexes were without statistical differences. Number of corpora lutea and count of implantation sites in females of all dose groups and in the control group were without statistical differences. Detailed histological examination was performed on reproduction organs and kidneys (target organs) of the males and female of the control and the highest dose group. Macroscopical and histopathological findings showed that Dusantox 86 did not cause pathological lesions in organs of treated animals. The sporadic presence of lesions in the animals of the high dose groups was similar to control groups.

CONCLUSION

The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:

in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The results of the reproduction/developmental screening study indicated that per os application of the test article Dusantox 86 didn’t show any significant changes in all used doses in comparison with the control animals:

in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

 

 

Effects on developmental toxicity

Description of key information

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February 2021 to 15 April 2021

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:

OECD guideline no. 414 “Prenatal Developmental Toxicity Study” of 25 June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Animal supply and acclimatisation
A total of 116 Sprague Dawley SD virgin female rats, 9 weeks old (200-225 g) were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy. The male rats used were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal husbandry
The rats were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the mated females were housed in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

The required amount of the test item was suspended in the vehicle to reach the required concentrations of 5, 25 and 100 mg/mL. The formulation was prepared up to weekly interval based on the stability data measurements. Concentrations were calculated and expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (ERBC Study no. A4101). A 28 hour stability at room temperature and a 8 day stability at +4°C were verified in the range from 5 to 100 mg/mL.
Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. The results were within the acceptability limits stated in ERBC protocols for suspensions.

Details on mating procedure:

The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Duration of treatment / exposure:

All animals were dosed from Day 3 through Day 19 post coitum.

Frequency of treatment:

All animals were dosed once a day.

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control Level

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low Level

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Medium Level

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Level

No. of animals per sex per dose:

Each group comprised 24 mated female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

Choice of the species, administration route and dose levels
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels were selected by the Sponsor.

Administration of test item
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Maternal examinations:

Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.

Body weight
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

Terminal studies
Euthanasia
All animals at termination were killed by carbon dioxide inhalation on Day 20 post coitum and necropsied as detailed below. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.

Necropsy
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.

Organ weight, fixation and preservation
From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to brain weight was calculated for each animal.

Thyroid histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes.

Ovaries and uterine content:

The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals found dead or killed during the study);
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Blood sampling:

On Day 20 post coitum, blood samples for thyroid hormones determination (approximately 1 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short time frame (e.g. two hours, if possible) on the morning of the day of necropsy.
Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300 µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.

Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOVX1610),
according to immunoanalytical methods validated at the Test Site:
– RV-T3/R/S-BKM/RIA-002 for T3
– RV-T4/R/S-BKM/RIA-002 for T4
– RV-TSH/R/S-IZO/RIA-002 for TSH
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).
Total number of samples: 96 × 2 aliquots
The aliquots were maintained at -20°C until transfer to the Test Site indicated below, where the immunoanalysis was carried out:

BIOVETIM
VetAgro Sup, aile 1 niveau 3
1 avenue Bourgelat
69280Marcy l’Etoile
France

Prior to shipment, the Principal Investigator was notified by e-mail.
Samples were labelled as follows: study number, animal number, group and sex, sampling date. Samples were transported in dry ice, by a dedicated courier and were subject to the latter’s standard insurance policy. In the first instance, only the aliquots A were despatched to the Test Site. The backup samples (aliquots B) will disposed of after Study Director’s authorisation and following Sponsor’s approval.
A Final Delegated Phase Report with the results of these analyses was sent to ERBC for inclusion in the Final Study Report. The results were presented as individual data (mean of duplicates, where applicable), group mean and group standard deviation. Evaluation of the data and the statistical analysis was performed in ERBC.

Fetal examinations:

Examination of foetuses
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination.
Skeletal and fixed-visceral examinationswere performed in all groups. Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Anogenital distance (AGD):
The AGD of each live foetuses was measured on Day 20 post coitum. The AGD was normalized to the cube root of foetal body weight collected on Day 20 post coitum. Individual and mean data was reported.

Statistics:

For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of theWilliams test. The criterion for statistical significance was
p<0.05. The mean values, standard deviations and statistical analysis were calculated from
the actual values in the computer without rounding off.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:

Pre impl. Loss % = [(no. of corpora lutea – no. of implantations) / no. of corpora lutea] x 100

Post-implantation loss was calculated as a percentage from the formula:

Post impl. Loss% = [(no. of implantations – no. of foetuses) / no. of implantations] x 100

Total implantation loss was calculated as a percentage from the formula:

Total impl. Loss % = [(no. of corpora lutea – no. of live foetuses) / no. of corpora lutea] x 100

Sex ratios of the foetuses were calculated as the percentage of males.
All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Historical control data:

No specified in the study report

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were detected throughout the study. A palpable subcutaneous mass was recorded in one lowdose female (no. 73) during the last day of study. This formation did not show microscopically changes. Other signs like hairloss or scabs were occasionally recorded during the study in single animals, with no correlation with the dose.
Thus they were considered as minor clinical signs and not related to the administration with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls (no. 31, 33) and one low dose female (no. 81).
The final number of females with live foetuses on gestation day 20 was of:
– 22 in the control group (0 mg/kg/day)
– 23 in the low dose group (50 mg/kg/day)
– 24 in the mid-dose group (250 mg/kg/day)
– 24 in the high dose group (1000 mg/kg/day)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effects were seen in food consumption throughout the study.

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

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

Serum levels of T3, T4 and TSH did not differ between treated and control groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
No effects were seen in thyroid weight between treated and control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in thyroid gland of females receiving 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at ≥ 50 mg/kg/day, when compared to controls. The microscopic observations in control and treated females were consistent with those known to occur spontaneously in untreated SD rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no differences in the number of implantations, corpora lutea and the intrauterine
deaths between control and treated groups.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.

Details on maternal toxic effects:

Treatment with the test item did not cause changes in body weight or food consumption at all dose levels when compared to controls throughout the study. In addition, the animals did not showtreatment-related clinical signs. The serumlevels of TSH, T3 and T4 hormones were comparable between control and treated groups. No treatment-related changes were noted at necropsy of females or at thyroid weight. The microscopic analysis of thyroid did not reveal findings at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

food consumption and compound intake

gross pathology

histopathology: neoplastic

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The body weights did not differ between control and treated groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live foetuses did not differ between control and treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The the percentage of males respect to females did not differ between control and treated groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter and body weights did not differ between control and treated groups.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related findings were recorded at external examination of foetuses. One single small foetus (body weight below 2.7g) was found in Group 3 as abnormality.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, thoracic/sacral vertebrae and the presence supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, the findings were considered unrelated to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A total of three major abnormalities were found in the study. Two were related to the heart (incomplete septum and/or persistent truncus arteriosus) and observed in one control foetus (foetus no. 10; Dam no. 9) and in one foetus of group 3 (foetus 10; Dam no. 125).
The other major abnormality was related to brain (extreme enlarged lateral ventricles) and it was found in one foetus of group 3 (Dam no. 139). These malformations occurred in single instances and in the absence of a dose dependency. Therefore, they were considered incidental and unrelated to treatment.
The incidences of foetuses or litters with minor abnormalities or variations did not suggest any test item effect.
The overall data confirm that there were no treatment-related findings at fixed visceral examination of foetuses.

Details on embryotoxic / teratogenic effects:

The number of corpora lutea, implantations, viable foetuses, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment-related effects were seen in the mean foetal weight. The external examination of foetuses including the measurement of the anogenital distance did not show differences in treated animals at all dose levels when compared to the control group.
No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

SUMMARY OF REPRODUCTION DATA – GROUP DATA

	Dosage Levels mg/kg/day
Observations	0	50	250	1000
No. Dams Inseminated No. Dams That Conceived Percent Dams Conceived No. Dams Died During Study No. Dams with Resorptions only No. of Litters Mean Absolute Weight Gain Total No. Corpora Lutea Mean No. Corpora Lutea/Pregnancy Total No. Implantation Mean No. Implants/Pregnancy Total No. Live Fetuses Mean No. Live Fetuses/Pregnancy Total No. Dead Fetuses Mean No. Dead Fetuses/Pregnancy No. Dams with Resorptions Total No. Resorptions Mean No. Resorptions/Pregnancy Preimplantation Loss (%) Post Implantation Loss (%) Mean Live Foetal Weight (g)	24 22 91.7 0 0 22 66.9 326 14.8 320 14.5 317 14.4 0 0.0 3 3 0.1 2.1 1.0 3.97	24 23 95.8 0 0 23 63.6 345 15.0 339 14.7 333 14.5 0 0.0 5 6 0.3 1.7 1.8 3.96	24 24 100.0 0 0 24 80.5 349 14.5 347 14.5 336 14.0 0 0.0 4 11 0.5 0.8 3.2 4.03	24 24 100.0 0 0 24 63.2 368 15.3 362 15.1 357 14.9 0 0.0 4 5 0.2 1.4 1.3 3.98

 

CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE

Interval: 0 – 20 Days Group Observation	1 (24)		2 (24)		3 (24)		4 (24)
	A	B	A	B	A	B	A	B
APPERANCE Scab(s) Presence if palpable mass	2 0	8.3 0.0	1 1	4.2 4.2	0 0	0.0 0.0	0 0	0.0 0.0

Key:

() = Number of animals alive at start of interval

A = Number of animals affected

B = Percent of animals with observations during interval

 

BODY WEIGHT (g) OF PREGNANT FEMALES – GROUP DATA

Group(s)		Day of Phase
		0!	3”	6	9	12	15	18	20
1	(n) Mean SD	22 274.20 19.12	22 291.48 19.07	22 301.78 20.33	22 317.60 21.07	22 336.15 22.98	22 355.70 23.20	22 399.69 28.91	22 436.66 31.84
2	(n) Mean SD	23 272.39 12.58	23 285.38 12.44	23 300.41 13.42	23 312.48 13.97	23 331.93 14.84	23 351.01 17.37	23 392.40 20.65	23 430.58 24.53
3	(n) Mean SD	24 272.90 16.51	24. 294.75 18.34	24 308.83 17.96	24 323.23 18.55	24 342.84 19.29	24 363.27 22.68	24 409.54 26.36	24 448.51 30.88
4	(n) Mean SD	24 273.76 12.25	24 292.71 15.88	24 303.76 14.66	24 316.81 14.18	24 334.43 17.86	24 355.05 18.86	24 397.54 24.80	24 434.91 30.01

Note: ! = Gestation phase; “ = Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is statistically different from control at p < 0.01

Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)

 

BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES – GROUP MEAN DATA

Group(s)		Day of Phase
		3	6	9	12	15	18	20
1	(n) Mean SD	22 5.762 2.126	22 3.431 2.144	22 5.274 2.985	22 6.184 1.561	22 6.518 1.651	22 14.662 2.579	22 18.485 2.957
2	(n) Mean SD	23 4.333 2.875	23 5.008 2.997	23 4.024 1.611	23 6.484 1.554	23 6.359 1.548	23 13.798 2.233	23 19.091 3.190
3	(n) Mean SD	24 7.284 1.749	24 4.6914 2.049	24 4.801 1.795	24 6.539 1.735	24 6.810 2.109	24 15.422 2.629	24 19.485 3.184
4	(n) Mean SD	24 6.319 2.705	24 3.683 1.848	24 4.350 1.814	24 5.871 2.015	24 6.875 1.897	24 14.162 2.729	24 18.685 3.493

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is statistically different from control at p < 0.01

Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)

°= mean daily body weight gain over the previous period starting from gestation day 0

 

FOOD CONSUMPTION° (g/animal/day) OF FEMALES – GROUP MEAN CAGE DATA

Group(s)		Day of Phase
		3	6	9	12	15	18	20
1	(n) Mean SD	9 22.47 2.50	9 25.08 1.70	9 26.59 1.99	9 26.85 1.89	9 27.61 2.53	9 29.97 3.20	9 30.58 2.99
2	(n) Mean SD	9 20.57 4.56	9 25.43 1.92	9 26.39 1.64	9 28.04 2.14	9 27.84 3.29	9 30.09 2.49	9 30.54 3.98
3	(n) Mean SD	9 23.96 1.37	9 25.99 1.70	9 27.30 1.28	9 27.94 1.38	9 29.05 1.79	9 31.70 2.06	9 32.29 2.58
4	(n) Mean SD	9 23.66 1.19	9 25.28 1.33	9 26.54 1.65	9 27.28 2.56	9 28.30 1.89	9 30.65 2.80	9 31.29 2.03

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is statistically different from control at p < 0.01

Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)

°= food consumed over the previous period starting from Day 0 post coitum

 

TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT AND ABSOLUTE WEIGHT GAIN (g) OF PREGNANT FEMALES – GROUP MEAN DATA

Group		Terminal body weight (g)	Gravid uterus weight (g)	Absolute weight gain# (g)
1	Mean SD N	427.77 32.18 22	86.72 15.79 22	66.85 18.32 22
2	Mean SD N	423.44 25.60 23	87.50 13.16 23	63.56 24.12 23
3	Mean SD N	439.63 30.86 24	86.28 20.79 24	80.45* 16.53 24
4	Mean SD N	426.77 30.71 24	89.84 17.13 24	63.18 19.95 24

# = Body weight at necropsy minus gravid uterus weight, minus body weight at Day 0 of pregnancy

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test; Williams test if group means are different from control at p < 0.05

 

THYROID HORMONE DETERMINATION ON DAY 20 POST COITUM – GROUP MEAN DATA

Parameter/units	Group	1	2	3	4
Triiodothyronine nmol/L	Mean SD n	1.310 0.185 24	1.298 0.190 24	1.276 0.174 24	1.287 0.205 24
Thyroxine nmol/L	Mean SD n	20.4 4.2 24	19.8 4.1 24	19.0 1.9 24	18.5 1.9 24
THYROID STIMULATING HORMONE ng/ml	Mean SD n	7.93 1.79 24	7.49 1.31 24	7.58 1.81 24	7.12 1.25 24

Controls from group(s): 1           Subgroup(s): 1

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

 

LITTER DATA AND SEX RATIO OF FEMALES – GROUP MEAN DATA

Group		Corpora Lutea	Implan-tations	Uterine Deaths			Viable Young			% males	Implantation loss (%)			Litter Weight (g)	Mean Foetal Weight (g)
															By sex		Combined
				Early	Late	Total	Total	M	F		Pre	Post	Total		M	F
1	Mean SD (n)	14.82 2.40 22	14.55 2.67 22	0.14 0.35 22	0.00 0.00 22	0.14 0.35 22	14.41 2.70 22	7.18 2.28 22	7.23 1.93 22	49.43 12.29 22	2.06 5.38 22	0.99 2.59 22	3.04 5.60 22	57.27 11.74 22	4.09 0.21 22	3.85 0.23 22	3.97 0.21 22
2	Mean SD (n)	15.00 2.30 23	14.74 2.30 23	0.22 0.52 23	0.04 0.21 23	0.26 0.54 23	14.48 2.33 23	6.87 2.01 23	7.61 1.90 23	47.26 11.23 23	1.67 3.92 23	1.76 3.59 23	3.41 5.10 23	57.30 9.46 23	4.05 0.29 23	3.89 0.22 23	3.96 0.24 23
3	Mean SD (n)	14.54 2.92 24	14.46 3.05 24	0.46 1.64 24	0.00 0.00 24	0.46 1.64 24	14.00 3.44 24	7.67 2.87 24	6.33 2.41 24	54.50 13.51 24	0.81 2.77 24	3.16 10.98 24	3.97 11.10 24	56.25 14.09 24	4.11 0.23 24	3.93 0.26 24	4.03 0.24 24
4	Mean SD (n)	15.33 3.13 24	15.08 2.95 24	0.21 0.51 24	0.00 0.00 24	0.21 0.51 24	14.88 2.91 24	7.71 2.61 24	7.17 2.51 24	51.94 13.65 24	1.41 3.32 24	1.31 3.21 24	2.70 4.48 24	58.96 10.91 24	4.07 0.21 24	3.90 0.20 24	3.98 0.19 24

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test; William’s test of group means are different from control at p < 0.05

 

ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA

MALES

Parameter/units	Group	1	2	3	4
ANOGENITAL DISTANCE (mm)	Mean SD n	4.30 0.26 22	4.24 0.30 23	4.36 0.28 24	4.37 0.25 24
Foetus weight (g)	Mean SD n	4.10 0.21 22	4.05 0.29 23	4.11 0.23 24	4.07 0.21 24
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g^1/3	Mean SD n	2.69 0.16 22	2.66 0.18 23	2.72 0.16 24	2.74 0.16 24

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA

FEMALES

Parameter/units	Group	1	2	3	4
ANOGENITAL DISTANCE (mm)	Mean SD n	2.54 0.53 22	2.48 0.60 23	2.53 0.55 24	2.69 0.50 24
Foetus weight (g)	Mean SD n	3.85 0.23 22	3.89 0.22 23	3.93 0.26 24	3.90 0.20 24
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g^1/3	Mean SD n	1.62 0.35 22	1.58 0.38 23	1.60 0.34 24	1.71 0.32 24

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

ABSOLUTE ORGAN WEIGHTS (g) – GROUP MEAN DATA

FEMALES

Organ: Brain	Controls from group: 1	Data homogenous by Bartlett’s test (Dunnett’s test)
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01	Control 24 1.969 0.105	2 24 1.944 0.083 0.065 0.081	3 24 1.976 0.104 0.065 0.081	4 24 1.967 0.078 0.065 0.081

Analysis of variance: F ratio = 0.52         Df = 3/92           F probability = 0.677

Note: a * indicates group mean is significantly different from control at level of significance shown.

Organ: Thyroid	Controls from group: 1	Data homogenous by Bartlett’s test (Dunnett’s test)
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01	Control 24 0.0241 0.0048	2 24 0.0241 0.0056 0.0031 0.0039	3 24 0.0222 0.0042 0.0031 0.0039	4 24 0.0215 0.0032 0.0031 0.0039

Analysis of variance: F ratio = 2.13         Df = 3/92           F probability = 0.100

Note: a * indicates group mean is significantly different from control at level of significance shown.

 

ORGAN WEIGHTS° TO BRAIN WEIGHT – GROUP MEAN DATA

FEMALES

Organ: Thyroid	Controls from group: 1	Data homogenous by Bartlett’s test (Modified t test)
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01	Control 24 1.228 0.248	2 24 1.241 0.299 0.164 0.223	3 24 1.122 0.193 0.133 0.180	4 24 1.092 0.165 0.126* 0.171

Analysis of variance: F ratio = 2.49         Df = 3/92           F probability = 0.064

Note: a * indicates group mean is significantly different from control at level of significance shown.

° = expressed as % organ to brain weight ratio

 

MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE – GROUP INCIDENCE

	-- Females --
Group: Number in group:	1 24	2 24	3 24	4 24
Skin             Subcutaneous mass(es)	0	1	0	0
Uterus             Not pregnant	2	1	0	0
Whole animal             No abnormalities detected	22	22	24	24

 

EXTERNAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Group	Organ	Cat	Observation(s)	No. Foetuses			No. Litters
				Observed	Affected	%	Observed	Affected	%
1	Whole foetus		No abnormalities detected	317	317	100.00	22	22	100.00
2	Whole foetus		No abnormalities detected	333	333	100.00	23	23	100.00
3	Whole foetus Whole foetus	AN	No abnormalities detected Small	336 336	335 1	99.70 0.30	24 24	23 1	95.83 4.17
4	Whole foetus		No abnormalities detected	357	357	100.00	24	24	100.00

 

SKELETAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Group	Organ	Cat	Observation(s)	No. Foetuses			No. Litters
				Obs	Aff	%	Obs	Aff	%
1	Forepaw(s) Forepaw(s) Pelvic girdle Ribs Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae	AN AN AN AN AN VA AN AN AN AN VA VA VA VA VA AN AN AN VA VA VA VA AN VA VA	Metacarpal(s) no ossification 4^th Metacarpal(s) incomplete ossification Pubis incomplete ossification Short Rudimentary Short 14^th Arch(es) incomplete ossification General incomplete ossification Hyoid no ossification Temporal incomplete ossification Nasal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification Hyoid incomplete ossification Interparietal incomplete ossification Asymmetrical ossification Fused No ossification 6^th Incomplete ossification 6^th Incomplete ossification No ossification 5^th Incomplete ossification 5^th Centrum asymmetrical ossification Centrum incomplete ossification Centrum dumb-bell shaped	165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165	68 1 1 4 1 6 8 2 17 6 1 2 18 16 49 6 1 28 51 1 38 32 1 19 1	41.21 0.61 0.61 2.42 0.61 3.64 4.85 1.21 1.30 3.64 0.61 1.21 10.91 9.70 29.70 3.64 0.61 16.97 30.91 0.61 23.03 19.39 0.61 11.52 0.61	22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22	19 1 1 3 1 4 4 1 8 4 1 2 10 11 16 5 1 13 19 1 16 14 1 10 1	86.36 4.55 4.55 13.64 4.55 18.18 18.18 4.55 36.36 18.18 4.55 9.09 45.45 50.00 72.73 22.73 4.55 59.09 86.36 4.55 72.73 63.64 4.55 45.45 4.55
2	Forepaw(s) Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae	AN AN VA AN AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN AN VA VA	Metacarpal(s) no ossification 4^th Rudimentary Short 14^th Arch(es) incomplete ossification Frontal incomplete ossification Hyoid no ossification Temporal incomplete ossification Interparietal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification Hyoid incomplete ossification Asymmetrical ossification No ossification 6^th Bipartite 5^th No ossification 5^th Incomplete ossification 6^th Incomplete ossification Incomplete ossification 5^th Centrum no ossification Centrum bipartite Centrum incomplete ossification Centrum dumb-bell shaped	171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171	58 3 3 5 1 15 1 33 1 13 9 11 17 2 43 41 3 53 1 2 15 1	33.92 1.75 1.75 2.92 0.58 8.77 0.58 19.30 0.58 7.60 5.26 6.43 9.94 1.17 25.15 23.98 1.75 30.99 0.58 1.17 8.77 0.58	23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23	20 3 3 5 1 9 1 16 1 6 6 10 8 1 16 18 2 19 1 2 9 1	86.96 13.04 13.04 21.74 4.35 39.13 4.35 69.57 4.35 26.09 26.09 43.48 34.78 4.35 69.57 78.26 8.70 82.61 4.35 8.70 39.13 4.35
3	Forepaw(s) Ribs Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae	AN AN AN VA AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN VA	Metacarpal(s) no ossification 4^th Short Rudimentary Short 14^th Arch(es) incomplete ossification Temporal incomplete ossification Hyoid no ossification Hyoid incomplete ossification Interparietal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification No ossification 6^th No ossification Asymmetrical ossification No ossification 6^th Incomplete ossification 6^th Incomplete ossification 5^th Incomplete ossification Centrum bipartite Centrum incomplete ossification	174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174	60 1 1 1 6 1 7 7 38 2 9 13 1 9 32 49 46 1 3 21	34.48 0.57 0.57 0.57 3.45 0.57 4.02 4.02 21.84 1.15 5.17 7.47 0.57 4.02 18.39 28.16 26.44 0.57 1.72 12.07	24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24	20 1 1 1 5 1 4 6 15 2 9 8 1 4 13 16 16 1 3 11	83.33 4.17 4.17 4.17 20.83 4.17 16.67 25.00 62.50 8.33 37.50 33.33 4.17 16.67 56.17 66.67 66.67 4.17 12.50 45.83
4	Forepaw(s) Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae	AN AN VA AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN VA VA	Metacarpal(s) no ossification 4^th Rudimentary Short 14^th Arch(es) incomplete ossification Temporal incomplete ossification Hyoid no ossification Interparietal incomplete ossification Hyoid incomplete ossification Supraoccipital incomplete ossification Parietal incomplete ossification Asymmetrical ossification Bipartite 5^th No ossification 6^th Incomplete ossification Incomplete ossification 5^th No ossification 5^th Incomplete ossification 6^th Centrum bipartite Centrum incomplete ossification Centrum dumb-bell shaped	183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183	43 6 2 3 1 9 43 12 16 3 3 3 23 1 45 31 53 2 19 2	23.50 3.28 1.09 1.64 0.55 4.92 23.50 6.56 8.74 1.64 1.64 1.64 12.57 0.55 24.59 16.94 28.96 1.09 10.38 1.09	24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24	16 5 2 2 1 7 17 9 11 3 2 3 9 1 18 17 17 2 11 2	66.67 20.83 8.33 8.33 4.17 29.17 70.83 37.50 45.83 12.50 8.33 12.50 37.50 4.17 75.00 70.83 70.83 8.33 45.83 8.33

 

FIXED VISCERAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Group	Organ	Cat	Observation(s)	No. Foetuses			No. Litters
				Obs	Aff	%	Obs	Aff	%
1	Abdomen Brain Heart Heart Heart Heart Kidneys Kidneys Testis Ureter	VA AN AN AN MA VA AN VA AN VA	Umbelical vein left Ventricle enlarged moderate Ventricle enlarged Atrium enlarged Septum incomplete Ventricle enlarged Ectopic Pelvic dilation slight Displaced Enlarged slight	152 152 152 152 152 152 152 152 152 152	1 1 1 1 1 2 2 1 10 7	0.66 0.66 0.66 0.66 0.66 1.32 1.32 0.66 6.58 4.64	22 22 22 22 22 22 22 22 22 22	1 1 3 1 1 3 2 1 9 4	4.55 4.55 13.64 4.55 4.55 13.64 9.09 4.55 40.91 18.18
2	Abdomen Abdomen Great vessels Heart Kidneys Kidneys Testis Thoracic cavity Ureter	VA VA VA AN AN VA AN VA VA	Umbelical vein left Haemorrhagic Long innominate artery Atrium enlarged Ectopic Pelvic dilation slight Displaced Haemorrhage Enlarged slight	162 162 162 162 162 162 162 162 162	2 2 1 4 4 1 5 2 5	1.23 1.23 0.62 2.47 2.47 0.62 3.09 1.23 3.09	23 23 23 23 23 23 23 23 23	2 2 1 2 3 1 2 2 5	8.70 8.70 4.35 8.70 13.04 4.35 8.70 8.70 21.74
3	Abdomen Abdomen Brain Great vessels Heart Heart Heart Heart Kidneys Kidneys Testis Testis Thoracic cavity Ureter Ureter	VA VA MA VA AN MA MA VA AN VA AN AN VA AN VA	Haemorrhagic Umbelical vein left Ventricles enlarged extreme Long innominate artery Atrium enlarged Septum incomplete Persistant truncus areriosus Pericardium Haemorragic Ectopic Pelvic dilation slight Malpositioned Displaced Haemorrhage Kinked moderate Enlarged slight	162 162 162 162 162 162 162 162 162 162 162 162 162 162 162	3 1 1 1 2 1 1 1 4 2 1 5 5 1 4	1.85 0.62 0.62 0.62 1.23 0.62 0.62 0.62 2.47 1.23 0.62 3.09 3.09 0.62 2.47	24 24 24 24 24 24 24 24 24 24 24 24 24 24 24	3 1 1 1 2 1 1 1 3 2 1 5 3 1 4	12.50 4.17 4.17 4.17 8.33 4.17 4.17 4.17 12.50 8.33 4.17 20.83 12.50 4.17 16.67
4	Abdomen Great vessels Heart Heart Kidneys Kidneys Testis Testis Thoracic cavity Thoracic cavity Ureter Ureter Ureter	VA VA AN VA AN VA AN AN AN VA AN AN VA	Haemorrhagic Long innominate artery Atrium enlarged Pericardium Haemorragic Ectopic Pelvic dilation slight Hypoplasia Displaced Haemorrhage Haemorrhage Enlarged moderate Kinked moderate Enlarge slight	173 173 173 173 173 173 173 173 173 173 173 173 173	7 3 6 2 1 1 1 10 2 1 1 1 3	4.05 1.73 3.47 1.16 0.58 0.58 0.58 5.78 1.16 0.58 0.58 0.58 1.73	24 24 24 24 24 24 24 24 24 24 24 24 24	5 2 4 1 1 1 1 9 2 1 1 1 3	20.83 8.33 16.67 47.17 4.17 4.17 4.17 37.50 8.33 4.17 4.17 4.17 12.50

 

Conclusions:

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.

Executive summary:

Study design

The effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were investigated in female Sprague Dawley SD rats during pregnancy and embryo-foetal development according to OECD guideline no. 414 “Prenatal Developmental Toxicity Study”. Females were in-house mated with sexually mature males of the same strain and then assigned to 4 groups of 24 females each as indicated below:

 

Group Number	Treatment (mg/kg/day)+	Level	Female Numbers (odd only)
1 2 3 4	0 50 250 1000	Control Low Medium High	1-47 49-95 97-143 145-191

+: in terms of test item supplied

 

The test item was suspended in 0.5 % methyl cellulose (0.5% MC) and administered by oral gavage at 50, 250 and 1000 mg/kg/day with the dose volume set at 10 mL/kg body weight.

The animals were treated throughout the gestation period from Day 3 up to Day 19 post coitum. Control females received the vehicle (0.5 % MC) at the same dose volume during the same treatment period.

 

Parameters assessed included clinical observations, body weight and food consumption during the in-life phase. Blood collection for hormones determination (T3, T4 and TSH) was performed on Day 20 post coitum before dispatch to necropsy.

 

All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination along with thyroid and brain weights. The ovaries were examined and the corpora lutea counted. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses.

 

Live foetuses were weighed, sexed and observed for external abnormalities. In addition, the anogenital distance (AGD) was recorded in all live foetuses.

 

Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

 

Histopathology of thyroid was performed in all groups.

 

Fate of females

All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls and one low dose female.

The final number of females with live foetuses on gestation Day 20 was as follows:

– 22 in the control group (0 mg/kg/day)

– 23 in the low dose group (50 mg/kg/day)

– 24 in the mid-dose group (250 mg/kg/day)

– 24 in the high dose group (1000 mg/kg/day)

 

Clinical signs

No treatment-related findings were recorded.

 

Maternal body weight and body weight gain

No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.

 

Food consumption

No treatment-related effects were seen in food consumption throughout the study.

 

Thyroid hormone determination

Serum levels of T3, T4 and TSH did not differ between treated and control groups.

 

Terminal body weight, uterus weight and absolute weight gain

Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.

 

Organ weight

No effects were seen in thyroid weight between treated and control groups.

 

Litter data and sex ratios

Litter data and sex ratios were unaffected by treatment.

 

Anogenital distance

No differences were seen in the anogenital distance between treated and control groups both in male and females foetuses.

 

Macroscopic and microscopic examinations

At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

 

External examinations of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Skeletal examinations of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Visceral examination of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Conclusion

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.

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:

K1

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

Prenatal Developmental Toxicity Study

Study design

The effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were investigated in female Sprague Dawley SD rats during pregnancy and embryo-foetal development according to OECD guideline no. 414 “Prenatal Developmental Toxicity Study”. Females were in-house mated with sexually mature males of the same strain and then assigned to 4 groups of 24 females each as indicated below:

 

Group Number	Treatment (mg/kg/day)+	Level	Female Numbers (odd only)
1 2 3 4	0 50 250 1000	Control Low Medium High	1-47 49-95 97-143 145-191

+: in terms of test item supplied

 

The test item was suspended in 0.5 % methyl cellulose (0.5% MC) and administered by oral gavage at 50, 250 and 1000 mg/kg/day with the dose volume set at 10 mL/kg body weight.

The animals were treated throughout the gestation period from Day 3 up to Day 19 post coitum. Control females received the vehicle (0.5 % MC) at the same dose volume during the same treatment period.

 

Parameters assessed included clinical observations, body weight and food consumption during the in-life phase. Blood collection for hormones determination (T3, T4 and TSH) was performed on Day 20 post coitum before dispatch to necropsy.

 

All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination along with thyroid and brain weights. The ovaries were examined and the corpora lutea counted. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses.

 

Live foetuses were weighed, sexed and observed for external abnormalities. In addition, the anogenital distance (AGD) was recorded in all live foetuses.

 

Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

 

Histopathology of thyroid was performed in all groups.

 

Fate of females

All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls and one low dose female.

The final number of females with live foetuses on gestation Day 20 was as follows:

– 22 in the control group (0 mg/kg/day)

– 23 in the low dose group (50 mg/kg/day)

– 24 in the mid-dose group (250 mg/kg/day)

– 24 in the high dose group (1000 mg/kg/day)

 

Clinical signs

No treatment-related findings were recorded.

 

Maternal body weight and body weight gain

No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.

 

Food consumption

No treatment-related effects were seen in food consumption throughout the study.

 

Thyroid hormone determination

Serum levels of T3, T4 and TSH did not differ between treated and control groups.

 

Terminal body weight, uterus weight and absolute weight gain

Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.

 

Organ weight

No effects were seen in thyroid weight between treated and control groups.

 

Litter data and sex ratios

Litter data and sex ratios were unaffected by treatment.

 

Anogenital distance

No differences were seen in the anogenital distance between treated and control groups both in male and females foetuses.

 

Macroscopic and microscopic examinations

At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

 

External examinations of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Skeletal examinations of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Visceral examination of foetuses

No treatment-related effects were seen at external examination of foetuses.

 

Conclusion

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.

 

Toxicity to reproduction: other studies

Additional information

No additional information

Justification for classification or non-classification

The above studies has been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the study was conducted to GLP and in accordance with an appropriate OECD Guideline. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results are considered to trigger no classification under the CLP Regulation (EC No 1272/2008)