Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 2006 - 5 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1100

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

EC Number:

309-912-6

EC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Cas Number:

101357-15-7

Molecular formula:

This is a UVCB substance. See section 1.2 for individual components.

IUPAC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: (P) 9 weeks
- Weight at study initiation: (P) Males: 316 - 358 g (mean 333.3 g); Females: 209 - 247 g (mean 226.5)

- Housing: Stainless mesh hanging type cages (195W x 325D x 180H mm; Tokiwa Kagaku Kikai Co., Ltd,) sterilised by autoclave were used and replaced more than once every two weeks (up to 15 days).
Gestation and lactation period of females: Polycarbonate cage (265W x 426D x 200H mm, Tokiwa Kagaku Kikai Co. Ltd.) sterilised by autoclave was used and replaced more than once a week (up to 8 days). Hardwood chips (Beta-Chip, Charles River Laboratories Japan, Inc.) sterilised by autoclave were used and replaced more than once a week (up to 8 days).
Number of animals: 1 male and 1 female during the mating period, 1 litter during the lactation period, and 1 animal during the other period including the quarantine and acclimation periods.

- Diet (e.g. ad libitum): ad libitum. Pellet diet for experimental animals (CRF-1), Oriental Yeast Co., Ltd., Lot. No.060411) sterilised by irradiation.
- Water (e.g. ad libitum): ad libitum tap water filtered through a 5 µm filter and disinfected by UV irradiation.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 25.2 °C
- Humidity (%): 48.4 - 66.5 % relative
- Air changes (per hr): 6 - 20 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light per day (7.00 to 19.00)

IN-LIFE DATES: From: To: 5 July 2006 - 3 September 2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Dose volume
The dose volume was set at 4 mL/kg, and the dosing volume for individual animals was calculated based upon the most recent measured body weight.

Preparation of dosing solutions - Method and frequency
Dosing solutions were prepared at least once every 10 days under UV protection light. The test substance was weighed for each concentration, and then mixed with Arachis oil in a mortar. The mixture was collected, when suspended homogeneously and further vehicle was added to give final concentrations. After stirring thoroughly, the solution was divided into polypropylene containers for each dosing day. The dosing solutions were preserved for up to 10 days in a refrigerator (actual temperature 4.7 - 7.0°C), shielded from light until each day of administration.

Details on mating procedure:

During the mating period, females cohabited on a one-to-one basis with males in the same group, day and night, for up to 14 days from the evening on day 14 (start of mating).
Vaginal smears were collected daily in the morning, from the day following the start of mating, and examined microscopically. The presence of vaginal plug or sperm in the vaginal smears confirmed copulation, and the day on which evidence of copulation was found was designated as day 0 of gestation. The following parameters were calculated.
(1) Days until copulation: Number of days from the start of mating to detection of copulation
(2) Number of oestrus stages without copulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Confirmation of the concentration
It was confirmed that the mean concentrations were within ± 10 % of the nominal concentration and C.V. values (from top, middle and bottom layers) were within 10 %. The criteria are as follows: concentration 97.3 - 105.2 %; C.V. 0.5 - 5.1 %.

Analysis method of the dosing solutions
Equipment
Spectrophotometer: U-3310 type (Hitachi, Ltd.)
Light source: Tungsten (w) lamp
Cell: 10 mm (silica glass)
Control: Dichloromethane (CH2Cl2)
Scan range: 300 nm - 700 nm
Scan speed: 300 nm/min
Slit width: 2 nm
Wave length: Absorbance (abs) at 562 nm


Standard solution
-Linear expression of a calibration curve was calculated using the measured concentration of the standard solution and its absorbance.
-Using the linear expression of the calibration curve, concentration of the test substance in the sample solution and dosing solution was calculated using the dilution factor of the sample solution.

Duration of treatment / exposure:

Males
The male animals were dosed for 42 days; 14 days before mating through the mating period to the day before necropsy.
Females
The female animals were dosed for 14 days before mating, during the mating period, gestation period, and delivery to day 3 of lactation (the date of delivery was designated as day 0 of lactation).

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were set according to the results of a 28-day repeated dose oral study (dose levels: 0, 15, 150 and 1000 mg/kg, five animals of both sexes at each group).

Examinations

Parental animals: Observations and examinations:

The following items were observed and examined. Day 0 is defined as the day of the first administration, while week 1 is defined as the period from day 0 to day 6. The day of copulation and parturition were designated as day 0 of gestation and day 0 post partum, respectively.


Clinical signs
The animals were observed twice a day for clinical signs (before and after dosing) during the treatment period and once in the morning during the other periods.


Bodyweight
The male animals were weighed on days 0, 3, 7, 14, 21, 28, 35, and 42. The female animals were weighed on days 0, 3, 7, and 14; days 0, 7, 14, and 20 of gestation and on days 0 and 4 of lactation.

Bodyweight gain was calculated on the basis of the start of dosing (day 0) for males and on day 0, day 0 of gestation, and day 0 of lactation for females.


Food consumption
Food consumption for males was measured on days 0 - 3, 3 - 7, 7 - 14, 28 - 35, and 35 - 42. Food consumption for females was measured on days 0 - 3, 3 - 7, and 7 - 14; days 0 - 7, 7 - 14, and 14 - 20 of gestation after copulation, and days 0 - 4 of lactation after delivery. Food consumption was not measured during the mating period.

Oestrous cyclicity (parental animals):

Reproductive performance test
Oestrous cycle
Vaginal smears were taken from females every morning from the start of the dosing period to the first day of mating period. Then mean oestrous cycle and incidences of irregular oestrous cycle were calculated.

Observation of parturition and lactation
All mated females were delivered naturally. For parturition, the dams were observed twice a day from day 21 to day 25 of gestation. The day when parturition was completed was designated as day 0 of lactation (day of completed delivery). Dams were observed every day for maternal behaviour, including lactation, nest building, and cannibalism until day 4 post partum.

Observation after lactation
The ovaries and uterus were extracted at necropsy and dams were examined for the number of corpora lutea and number of implantation sites. The following indices were calculated:
(1) Gestation length: Number of days from day 0 of gestation to the day of parturition
(2) Gestation index (%): (Number of females with live offspring / number of pregnant females) x 100
(3) Implantation index (%): (Number of implantation sites / number of corpora lutea) x 100
(4) Birth index (%): (Number of offspring born alive / number of implantation sites) x 100

Litter observations:

Observation of offspring
On day 0 of lactation, litters were examined for the number of offspring (live or stillborn); newborns were examined for sex, and external anomalies. After that, all litters were observed daily for clinical signs and mortality. All live offspring were examined for external anomalies including those in the oral cavity on day 4 of post partum.

Body weight
All offspring were weighed individually on days 0 and 4 of lactation. Body weight gain was calculated on the basis of the body weight measured on day 0 post partum.

Postmortem examinations (parental animals):

Organ weights
Relative organ weights (versus body weight ratio) were calculated from body weights on the day of necropsy.


Necropsy
The males on day 42, and delivered females on day 4 of lactation were euthanised by exsanguination from the abdominal aorta under anaesthesia with an intraperitoneal injection of a pentobarbital sodium and then subjected to necropsy. One dam that exhibited total litter loss was necropsied immediately.


HistopathologicaI examination
Organs and tissues as described below were removed from all animals. The sampled organs/tissues were preserved after fixing them in 10 v/v % neutral phosphate-buffered formalin solution. The testes and epididymis were fixed first with Bouin's solution, and then preserved in 10 v/v % neutral phosphate-buffered formalin solution.
Testes
Epididymis
Prostate (ventral lobe)
Seminal vesicle (including coagulating gland)
Ovaries
Organs/tissues of animals showing gross lesions

The testes and epididymis in the males, the ovaries in the females of the control group and 1000 mg/kg group, and organs/tissues of all animals showing gross lesions were prepared by haematoxylin and eosin staining the specimens and then they were microscopically examined.
However, a histopathological examination on the changes attributed to the colour of the test substance was not performed as the NOAEL was already confirmed in a 28-day repeated dose oral study. The relative organs/tissues were only preserved.

Postmortem examinations (offspring):

The animals were euthanised in the same manner as the parental animals and subjected to necropsy. Concerning the changes attributed to the colour of the test substance, three representative animals were preserved in 10 v/v % neutral phosphate-buffered formalin solution. In addition, three random samples from the animals in the control group were preserved in the same manner as described previously.
Dead offspring, except for those that died due to cannibalism, were immersed and fixed in 10v/v % neutral phosphate-buffered formalin solution following the necropsy.

Statistics:

Offspring data were analysed on the basis of litter mean values.
Statistical significance of the metric data was analysed by multiple comparison tests. First, homogeneity of the data was tested by Bartlett's test. If the variance was homogeneous, one-way analysis of variance was performed for statistical comparison. If the variance was heterogeneous, the Kruskal-Wallis test was used. When a significant inter-group difference was found, Dunnett’s (homogeneous) or Dunnett-type (heterogeneous) multiple comparison test was used. For some parts of the data, the Kruskal-Wallis test was applied first, and when a significant inter-group difference was found, Dunnett-type multiple comparison was used. The numerical data in the histopathological examination was analyzed by Wilcoxon's rank sum test. Other numerical data was analysed by Fisher's exact probability test. The significant level of 5 % was set for all statistical analysis.

Statistical analysis was performed on the items listed below. The analysis was not performed on clinical signs or necropsy findings.

Multiple comparison test:
Body weight, body weight gain, food consumption, organ weight, number of corpora lutea, number of implantations and number of offspring (live or stillborn).

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of oestrus stages without copulation, mean oestrous cycle, gestation length, implantation index, birth index, live birth index, incidence of external anomalies and viability index on day 4.

Wilcoxon's rank sum test:
Histopathological examination.

Fisher's exact probability test:
Incidences of irregular oestrus cycle, copulation index, fertility index, gestation index, sex ratio (male/female), and incidence of dams with externally anomalous offspring

Reproductive indices:

(1) Copulation index (%): (Number of animals with successful copulation / Number of animals examined) x 100
(2) Fertility index (%): (Number of pregnant animals / Number of animals with successful copulation) x 100

Offspring viability indices:

(1) Live birth index (%): (Number of offspring born alive/ Total number of offspring born) x 100
(2) Viability index on day 4 (%): (Number of offspring alive on day 4 / Number of offspring born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See below

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Clinical signs
No deaths occurred in both sexes in the treated groups.
Blackish faeces were observed in both sexes in all treated groups throughout the treatment periods. Coloured fur was also observed sporadically in some males receiving 1000 mg/kg/day, and females receiving doses of and above 150 mg/kg/day.


Necropsy findings
Abnormal contents in the stomach were observed in 2 females receiving 150 mg/kg/day in addition to 2 males and 4 females receiving 1000 mg/kg/day. A blackish patch in the anterior stomach mucosa was observed in 4 males and 2 females receiving 150 mg/kg/day and 8 males and 8 females receiving 1000 mg/kg/day.
A pinkish patch in the adipose tissue was observed in 9 males and all females receiving 150 mg/kg/day, as well as all animals of both sexes receiving 1000 mg/kg/day. No other particular abnormalities were noted.


HistopathologicaI findings
No treatment-related changes were noted, though atrophy of the seminiferous tubule in the testis (unilateral) was noted in one male in the 1000 mg/kg group; however, it was not considered to be treatment-related because there was only one incidence and it is a spontaneous change in rats.


Reproductive performance test
All mated animals including those in the control group were successfully copulated, and the percentages of both copulation and fertility indices were 100 % in each group


Observation of parturition and lactation
There were no statistically significant differences observed in gestation length, number of corpora lutea, number of implantations, implantation index, gestation index, or birth index with those of the control group. All dams showed no abnormalities in their delivery and nursing.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of overt toxicity noted.

Results: F1 generation

General toxicity (F1)

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:

effects observed, treatment-related

Description (incidence and severity):

See below

Histopathological findings:

not examined

Details on results (F1)

Observation of offspring
Significant differences in the sex ratio were observed in offspring and live offspring (on days 0 and 4 of post partum) in the 150 mg/kg and 1000 mg/kg groups. This variance is considered to be incidental because the number of female offspring in the control group was larger than males, while the number of male offspring in the 150 mg/kg and 1000 mg/kg groups was slightly larger than females. There were no differences in the numbers of offspring and live offspring between the control group and the 150, 1000 mg/kg groups.


Necropsy findings
At the necropsy of live offspring on day 4 of post partum, abnormal contents in the stomach were observed in all animals of both sexes receiving 1000 mg/kg/day (one male had a dark-reddish content, and the rest had pinkish milk). A pinkish patch in the adipose tissues was observed in all animals of both sexes receiving 150 mg/kg/day and 1000 mg/kg/day. A dark-reddish patch in the lung was observed in one male receiving 1000 mg/kg/day; however, it was judged incidental with no relationship with the test substance in consideration of its incidence.

At the necropsy of dead offspring, abnormal contents in the stomach was observed in one male receiving 150 mg/kg/day, and a pinkish patch in the adipose tissue was observed in one male receiving 1000 mg/kg/day.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of overt toxicity noted.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1 Parental Necropsy Organ Findings - Summary

Sex	Male				Female
Dose (mg/kg)	0	15	150	1000	0	15	150	1000
No. of animals	12	12	12	12	12	12	12	12
Stomach Abnormal contents Tinction*	0 0	0 0	0 4	2 8	0 0	0 0	2 2	4 8
Adipose tissue Tinction	0	0	9	12	0	0	12	12

 *Forestomach, mucosa

Table 2 Offspring Necropsy Findings

Scheduled Sacrifice

Dose (mg/kg)	Findings	Scheduled Sacrifice
		Day 4
		M	F
0	No. examined No abnormality	77 77	108 108
15	No. examined No abnormality	79 79	90 90
150	No. examined Adipose tissue tinction (pink)	99 99	82 82
1000	No. examined Adipose tissue tinction (pink) Stomach abnormal contents (pink) Stomach abnormal contents (dark red) Lung (dark reddish change)	94 94 93 1 1	85 85 85 0 0

 Found Dead

Dose (mg/kg)	Findings	Dead
		Day 0		Day 1		Day 2		Day 3		Day 4
		M	F	M	F	M	F	M	F	M	F
0	No. examined No abnormality
15	No. examined No abnormality Cannibalism				1   1			1 1	2 2
150	No. examined Stomach abnormal contents (pink) Cannibalism						1   1			2 1 1
1000	No. examined No abnormality Adipose tissue tinction (pink) Cannibalism	1 1				1     1			1     1	1   1	1 1

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL for parental animals and neonates is considered to be 1000 mg/kg for reproductive and developmental toxicity.

Executive summary:

The effects of the test substance on reproduction/developmental toxicity were examined in accordance with OECD Guideline for the Testing of Chemicals Number 421 and US EPA Health Effects Test Guidelines OPPTS 870.1000 and OPPTS 870.1100.

During the study, the test material was administered orally to rats at dose levels of 15, 150, and 1000 mg/kg by oral gavage in arachis oil , with 12 animals per sex receiving each dose. Male animals were dose for 14 days before mating then throughout the mating period up until the day before necropsy (42 days). Female animals were dosed for 14 days before mating, during the mating period, gestation period and delivery up until day 3 of lactation.

Parental observations included blackish faeces in all animals of both sexes throughout the dosing periods at all concentrations. At the necropsy, abnormal contents in the stomach, a blackish patch in the anterior stomach mucosa and a pinkish patch in the adipose tissue were observed in all animals of both sexes receiving 150 and 1000 mg/kg/day. These changes had a close resemblance to those shown in a 28-day repeated dose oral study, which was conducted prior to this study. They are considered attributed to the colour of the test substance, suggesting no toxicological significance. No abnormalities were noted in the oestrus cycle, copulation index, fertility index, required days until copulation, number of oestrus stages without copulation, gestational period, number of corpora lutea, number of implantation, number of offspring, implantation index, delivery index, or birth index in any treated groups. Therefore, the test substance was judged to have no effects on reproductive functions.

 

Effects on the offspring: abnormal contents in the stomach and pinkish patch in the adipose tissue were observed in both sexes in the 150 and 1000 mg/kg groups. These changes were considered attributed to the colour of the test substance because the dams showed almost the same changes. Moreover, no anomalies were observed in external, clinical signs or survival index. Therefore, it was judged that the changes attributed to the colour of the test substance had no toxicological significance on the offspring (next generation).

 

As described above, although the changes attributed to the colour of the test substance were noted in parental animals and neonates, no changes suggestive of toxicity were noted up to the highest dose tested, 1000 mg/kg/day. Thus, the NOAEL under the conditions of this study for parental animals and neonates was considered to be 1000 mg/kg for reproductive and developmental toxicity.