Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Namiki (2007)

Under the conditions of the study, the NOAEL for parental animals and neonates is considered to be 1 000 mg/kg for reproductive and developmental toxicity.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1100

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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.

Dose / conc.:

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

Dose / conc.:

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

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).

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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.

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

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.

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.

Reproductive effects observed:

not specified

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

 

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.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: 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:

The key study was conducted in compliance with GLP and in accordance with the OECD Guideline for the Testing of Chemicals Number 421 and US EPA Health Effects Test Guidelines OPPTS 870.1000 and OPPTS 870.1100. It has been assigned a reliability score of 1 according to the principles for assessing data quality as set out by Klimisch (1997).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Namiki (2007)

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the test material was administered orally to rats at dose levels of 15, 150, and 1 000 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 1 000 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 1 000 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, 1 000 mg/kg/day. Thus, the NOAEL under the conditions of this study for parental animals and neonates was considered to be 1 000 mg/kg for reproductive and developmental toxicity.

Effects on developmental toxicity

Description of key information

Namiki (2007)

Under the conditions of the study, the NOAEL for parental animals and neonates is considered to be 1 000 mg/kg for reproductive and developmental toxicity.

Van Tuyl (2007)

Under the conditions of the study, the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1 000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2007 - 11 July 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 10 - 12 weeks
- Weight at study initiation: 214 - 311 g
- Housing: 5 animals per cage (pre-mating); 1 male and 1 female per cage (mating); individually (post-coitum)
- Diet: pelleted rodent diet (SM R/M-Z, SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 24.0 ºC
- Humidity (%): 42 - 85 %
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 9 May 2007 - 11 July 2007

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: formulations of test material in vehicle were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. Dosing solutions were stored at ambient temperature prior to use.
- Dose volume: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples: duplicate samples (approximately 250 or 500 mg) were accurately weighed into volumetric flasks of 25, 100 or 250 mL. For determination of accuracy, samples were taken at 50 % height or at 90 %, 50 % and 10 % height. The latter set of samples was also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50 % height. The flasks were filled up to the mark with methanol. The solutions were ultrasonicated for 10 minutes. The solutions were further diluted with methanol to obtain concentrations within the calibration range.

- Analytical conditions:
- Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
- Detector: Dual λ Absorbance Detector 2487 (Waters)
- Column: 100 mm x 4.6 mm i.d. Symmetry Shield RP-18, dp = 3.5 µm (Waters)
- Injection volume: 100 µL
- Mobile phase: 0.1 % H3PO4 in 70/30 (v/v) methanol/Milli-Q water
- Flow: 0.6 mL/min
- UV detection: 290 nm

- Sample Injection: calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection

- Calibration curves: a calibration curve was constructed using four concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation was > 0.99.

Details on mating procedure:

Females were caged together with males on a one-to-one basis during the mating period.

Duration of treatment / exposure:

From day 6 to day 19 post-coitum, inclusive.

Frequency of treatment:

Once daily 7 days per week, approximately the same time each day with a maximum of 5 hours and 12 minutes difference between the earliest and latest dose.

Duration of test:

4 weeks.

Remarks:

Doses / Concentrations:
0, 15, 150 and 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 females (0, 15, 150 mg/kg/day); 25 females (1000 mg/kg/day).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. Four groups of 6 females were exposed to 0, 15, 150 and 1000 mg/kg/day for days 6 to 19 post-coitum inclusive by oral gavage. These dose levels were based on a 28-day toxicity study in which rats were exposed to the same dose levels. Under the conditions of the study, no mortality occurred and there were no treatment related findings in clinical signs, body weights and food consumption. There were no toxicologically relevant changes noted during macroscopic and microscopic examination and so 0, 15, 150 and 1000 mg/kg/day were selected as dose levels for the main developmental study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for viability/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from day 0 post-coitum onwards. The time of onset, degree and duration was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3 and 6 - 20 (daily) post-coitum

FOOD CONSUMPTION: Yes
- Time schedule: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 post-coitum
- Organs examined: Blood was collected for haematological examination. External thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. Liver and spleen were collected and fixed in 10 % buffered formalin. Each ovary and uterine horn was dissected and examined. Furthermore, the female genital tract including placentas was preserved in 10 % buffered formalin for possible histological examination.

HAEMATOLOGICAL EXAMINATION: Blood samples were collected under isoflurane anaesthesia. Blood samples were taken from the aorta and examined for the following parameters: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.

ORGAN WEIGHTS: The weight of the liver, spleen and terminal body weight were recorded from the surviving females on the scheduled day of necropsy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of live and dead fetuses: Yes
- Number and distribution of embryo-fetal deaths: Yes
- Number of former implantation sites: Yes

Fetal examinations:

- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes: (al per litter)
- Skeletal examinations: Yes: (all per litter)
- Head examinations: Yes: (half per litter)

Statistics:

Where variable followed a normal distribution, the Dunnett-test was applied. For non-normalised data, the Steel-test was applied. The Fisher Exact-test was applied to frequency data. In all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data.

Indices:

Pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / Number of corpora lutea] x 100

Post-implantation loss = [(Number of implantation sites - number of live fetuses) / Number of implantation sites] x 100

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- Mortality; one animal treated at 1000 mg/kg was sacrificed on day 7 post-coitum, based on clinical signs and body weight loss (-14%). This death was not considered to be related to treatment, as the animal had bitten through the plastic gavage tube during dosing on day 6 post-coitum. At necropsy, blue discolouration of the gastro-intestinal tract was observed. This animal had 17 normally developing fetuses (8 in the right uterus horn, and 9 in the left horn) and 19 corpora lutea. An additional animal was added to the study to replace this animal. No other mortality occurred.

- Clinical signs: One animal treated at 1000 mg/kg showed rales from days 14 to 18 post-coitum. Black discolouration or staining of faeces and different parts of the body was noted at 150 mg/kg and 1000 mg/kg in a dose dependent manner. This discolouration was related to the colour of the test material and was not considered to be of toxicological relevance. One animal showed alopecia, which was considered to be unrelated to treatment as this was an isolated finding.

- Body weights: Body weights and body weight gain of treated animals remained in the same range as the controls over the 4-week study period.

- Food consumption: Food consumption before or after allowance for body weight was similar between treated and control animals.

- Haematology: No toxicologically relevant findings were noted. A slight increase in reticulocytes was noted in females treated at 1000 mg/kg. No other treatment related findings were noted. As the slight increase in reticulocytes was an isolated finding, and as no other erythrocyte parameters were affected the toxicological relevance of this finding was doubted.

- Pathology: Reddish discolouration in parts of the abdominal adipose tissue was noted in animals dosed at 150 and 1000 mg/kg. Black discolouration of the gastro-intestinal tract was noted in one 1000 mg/kg animal. Furthermore, two animals dosed at 1000 mg/kg showed black discolouration of the skin at necropsy. This discolouration was considered to be due to the colour of the test material and of no toxicological relevance. Other findings noted among control and treated animals (including uterus filled with fluid, alopecia, adhesion of the placenta) were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.

- Organ weights: Liver and spleen weights of treated animals were considered to be similar to those of control animals.

- Histopathology: There were no treatment related findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size: A slight reduction in the number of viable fetuses was noted in animals treated at 1000 mg/kg; however, this was not statistically significant as the value was well within the historical control range.

- Sex ratio: No treatment-related effect on the sex ration was noted.

- External malformations and variation: There were no test material related external malformations. No external developmental variations were observed in any fetuses during the study. Polydactyly was noted was noted in one fetus from the group dosed at 1000 mg/kg and also in one fetus in each the control and the 15 mg/kg dose groups. The incidence of polydactyly did not indicate a relation to treatment with the test material.

- Visceral malformations and variations: Situs inversus affecting all internal organs in one fetus of the 15 mg/kg group, and enlarged right atrium of a fetus in the 1000 mg/kg group were the only findings noted and were considered spontaneous in origin and hence not related to treatment with the test material. Soft tissue developmental variations noted in the test material treated groups were right subclavian originating from the aortic arch, accessory spleen, thymus extended into neck region, and accessory liver lobule. These findings were not considered test material related because the mean litter proportions of these variations were not dose related.

- Skeletal malformations and variations: Malformations that were noted during the study consisted primarily of bent limb bones that were noted in one fetus of the 1000 mg/kg group and five fetuses of the control group. One fetus in the 15 mg/kg group showed sternoschisis. These findings were not attributed to the test material due to occurrence in single fetuses and/or the absence of a dose relationship. In the control group one fetus had a vertebral centra anomaly in which the half of a thoracic centrum was absent.
There was a statistically significant decrease in the mean litter proportion of fetuses with reduced ossification of the skull, resulting in mean litter proportions of skeletal and total variations in the 1000 mg/kg group that were statistically significantly decreased. The values of reduced ossification of the skull were 19.9 %, 22.4 %, 18.8 % and 10.3 % in the control, 15, 150 and 1000 mg/kg groups respectively. These values indicate an advanced ossification of skull bones in the 1000 mg/kg group compared to the other groups. No patterns of advanced ossification were observed for the ossification of cervical centrum no. 1, vertebral arches, ribs and sternebra in the 1000 mg/kg group. Although a relationship to treatment cannot be dismissed, a decrease in the number of variations in fetuses that indicate a possible advancement in ossification of the skull is not considered an adverse developmental effect.
Other skeletal developmental variations observed in the test material groups were 14th rudimentary ribs, unossified hyoid, 7th cervical ribs, bent ribs, unossified sternebra nos. 5 and/or 6, slightly to moderately malaligned sternebra(e), 14th full ribs, unossified pubis, tarsals ossified, 25 presacral vertebrae and unossified vertebral centra. All these variations occurred at similar frequencies in the control, and/or occurred infrequently, and/or in a manner that was not dose related.

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 adverse test material related effects were observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

Formulation Analysis Results

The concentrations analysed in the formulations of the groups dosed at 15 and 1000 mg/kg were between 87 % and 100 % of target, which was as expected based on the recoveries of the procedural recovery samples (86 - 100 %). The accuracies of the 150 mg/kg formulations were slightly higher (108 - 116 %) but still considered acceptable. The 15 and 1000 mg/kg formulations were homogenous and stable for at least 5 hours when stored at room temperature. No test material was detected in the control group formulations.

Table 1: Summary of Fetuses and Litters with Malformations

	Dose group	Fetuses				Litters
		0	15	150	1000	0	15	150	1000
No. examined externally		337	358	305	316	23	24	21	23
Polydactyly		0	0	0	1	0	0	0	1
No. examined viscerally		337	358	305	316	23	24	21	23
Heart - atrium enlarged		0	0	0	1	0	0	0	1
Microphthalmia		1	0	0	0	1	0	0	0
Situs Inversus		0	1	0	0	0	1	0	0
No. examined skeletally		337	358	305	316	23	24	21	23
Bent limb bone(s)		5	0	1	0	2	0	1	0
Vertebral centra anomaly		1	0	0	0	1	0	0	0
Polydactyly		1	1	0	0	1	1	0	0
Sternoschisis		0	1	0	0	0	1	0	0
Total no. with malformations
External		0	0	0	1	0	0	0	1
Soft tissue		1	1	0	1	1	1	0	1
Skeletal		7	2	1	0	4	2	1	0
Combined		8	3	1	2	5	3	1	2

Conclusions:

Under the conditions of the study, the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1000 mg/kg bw/day.

Executive summary:

The prenatal developmental toxicity of the test material was determined in accordance with standardised guidelines OECD 414, EU Method B.31 and EPA OPPTS 870.3700. During the study, mated female Wistar rats were assigned to four dose groups, containing 24 animals (in groups dosed at 0, 15 and 150 mg/kg) and twenty five animals (in the group dosed at 1000 mg/kg), respectively. The test material was administered once daily by gavage from day 6 to 19 post-coitum. The rats of the control group received the vehicle, propylene glycol, alone. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Under the conditions of the study, no maternal mortality was observed in any of the dose groups. A slight increase in reticulocytes was noted in females treated at 1000 mg/kg. No other treatment related findings were noted. As the slight increase in reticulocytes was an isolated finding, and as no other erythrocyte parameters were affected, the toxicological relevance of this finding was doubted. No reproductive toxicity was observed during the study and no developmental toxicity was observed.

Based on the results in this prenatal developmental toxicity study the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1000 mg/kg bw/day.

Reference 2

Endpoint:

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.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Health Effects Test Guideline, OPPTS 870.1100

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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

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.

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.

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

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.

Dose / conc.:

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

Dose / conc.:

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

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).

Maternal 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 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 day 0, day 0 of gestation, and day 0 of lactation for females.


Food consumption
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.

Ovaries and uterine content:

There were no statistically significant differences observed in the number of corpora lutea, number of implantations, implantation index, gestation index, or birth index with those of the control group.

Fetal examinations:

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.

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

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

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs
No deaths occurred in the treated groups.
Blackish faeces were observed in all treated groups throughout the treatment period. Coloured fur was also observed sporadically in some females receiving 150 mg/kg/day or higher.


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


HistopathologicaI findings
No treatment-related changes were noted.


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 the dam animals showed no abnormalities in their delivery and nursing.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

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 test material related effects were observed at this dose.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of this study, the NOAEL for parental animals and neonates was 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.

The material was administered orally to SD 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: blackish faeces were observed 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 is 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 at doses up to, and including, 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.

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:

The key study was conducted in compliance with GLP and in accordance with the OECD Guideline for the Testing of Chemicals Number 414, EU Method B.31 and US EPA OPPTS 870.3700. It has been assigned a reliability score of 1 according to the principles for assessing data quality as set out by Klimisch (1997).

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

Namiki (2007)

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The material was administered orally to SD rats at dose levels of 15, 150, and 1 000 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: Blackish faeces were observed 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 1 000 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 1 000 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 is 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 at doses up to, and including, 1 000 mg/kg/day. Thus, the NOAEL, under the conditions of this study, for parental animals and neonates was considered to be 1 000 mg/kg for reproductive and developmental toxicity.

 

Van Tuyl (2007)

The prenatal developmental toxicity of the test material was determined in accordance with standardised guidelines OECD 414, EU Method B.31 and EPA OPPTS 870.3700. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, mated female Wistar rats were assigned to four dose groups, containing 24 animals (in groups dosed at 0, 15 and 150 mg/kg) and twenty five animals (in the group dosed at 1 000 mg/kg), respectively. The test material was administered once daily by gavage from day 6 to 19 post-coitum. The rats of the control group received the vehicle, propylene glycol, alone. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Under the conditions of the study, no maternal mortality was observed in any of the dose groups. A slight increase in reticulocytes was noted in females treated at 1 000 mg/kg. No other treatment related findings were noted. As the slight increase in reticulocytes was an isolated finding, and as no other erythrocyte parameters were affected, the toxicological relevance of this finding was doubted. No reproductive toxicity was observed during the study and no developmental toxicity was observed.

Based on the results in this prenatal developmental toxicity study the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1 000 mg/kg bw/day.

Justification for classification or non-classification

In accordance with the criteria set out in Annex I of Regulation No. 1272/2008, the test material does not require classification for toxicity to reproduction or developmental effects. There is no evidence of adverse effects on sexual function and fertility or on development of offspring, caused by exposure to the test substance.