Trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-May-2012 to 26-Nov-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well planned GLP and OECD guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 295 to 329 g (males) and 178 to 213 g (females)
- Identification: Cage card and individual animal number (ear tattoo).Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values for both temperature and humidity were outside these ranges but were transient variations and were considered not to have any influence on the study and, therefore, these data are not reported but retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) (batch/lot nos. 02105120601 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Route of administration:

oral: gavage

Vehicle:

other: Highly purified water (ELGA labwater)

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Acid Brown 425 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were divided into daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored in refrigerator (5 ± 3 °C) in brown bottles in daily aliquots.

Based upon the results of stability analyses performed within the Harlan Laboratories non-GLP study D51147 (Dose Range-Finding Study for a Reproduction/Developmental Toxicity Screen¬ing Test in the Han Wistar Rat), dose formulations were stable for at least one week in refrigerator.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (group 1, control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D51147, using dose levels of 0, 100, 300 and 1000 mg/kg/ day. No adverse toxic effects were found at any dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (group 1), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the responsible person for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS following an analytical procedure developed at Harlan Laboratories study D51160. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

RESULTS

The application formulations investigated during the study were found to comprise Acid Brown 425 in the range of 96.4% to 113.1% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Acid Brown 425 in the preparations was approved because single results found did not deviate more than 1.1% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature or eight days refrigerated due to recoveries which met the variation limit of ±10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Acid Brown 425 and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Minimum 4 weeks (males) or approximately 7 weeks (females)

Frequency of treatment:

Once daily

Details on study schedule:

MALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days of treatment

FEMALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Necropsy: On day 5 post partum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of Acid Brown 425 on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and lactation periods (females) including the day before scheduled necropsy.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Positive control:

No

Parental animals: Observations and examinations:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

The litters were examined for litter size, live births, live births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY

Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

A histopathology peer review was performed and the results included in the histopathology phase report.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All pups except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following paramters were calculated: fertility indices, mean precoital time, post-implantation losses and mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pups sex ratio and viability indices.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Black stained faeces in all test item-treaded animals ; this was due to the staining proprerties of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction in food consumption and body weight/body weight gain in males treated at 300 and 1000 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduction in food consumption and body weight/body weight gain in males treated at 300 and 1000 mg/kg bw/day

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:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

1. IN-LIFE DATA

VIABILITY/MORTALITY

All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS

Black colored feces were recorded in males and females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item.
In one male (no. 35) in the high dose group a wound in the neck region was recorded for a few days in the pairing period. In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in males or females at any dose level.

FOOD CONSUMPTION OF MALES

At 1000 mg/kg body weight/day, mean food consumption of males was statistically significantly decreased between days 1 - 14 in the pre-pairing period. This was considered to be a test item-related effect.

No effect on food consumption of males was observed in the 300 and 100 mg/kg body weight/day dose groups.


FOOD CONSUMPTION OF FEMALES

There were no effects on food consumption of females at any dose level.

BODY WEIGHTS AND BODY WEIGHT GAIN OF MALES

At 1000 and at 300 mg/kg body weight/day, statistically significantly reduced body weights were noted in males from day 11 until day 14 of the pre-pairing period and throughout the pairing period.

At 1000 and 300 mg/kg body weight/day, the mean body weight gains of males were statistically significantly reduced from early pre-pairing period until the end of this period. At 300 mg/kg body weight/day, the body weight gain was statistically significantly lower on certain days during the pairing period too (on Days 4-8 and 11, 12) but without toxicological relevance. The body weight gains were statistically significantly reduced and showed a dose dependent pattern in the pre-pairing period beginning from Day 2 and Day 3 of treatment in groups 4 and 3, respectively. The values were lower than in controls and in the low dose group, therefore the reduced body weight gains in the pre-pairing period were considered to be test item-related effects in groups 3 and 4.

The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day were respectively: +16%, +15%, +12% and +10% during the pre-pairing period (percentages refer to the body weight gain within the period).

BODY WEIGHTS AND BODY WEIGHT GAIN OF FEMALES

No test item-related effects on body weights and body weight gain of females were observed at any dose level.

2. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

No effect on mating performance or fertility was observed at any dose level.

Mating of all test item-treated females was recorded during the first pairing period. No mating was detected for one control female (no. 51) but it was pregnant and delivered pups. The precoital time was not affected by the treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 5.0 (3), 4.1 (3), 2.4 (3) and 4.4 (3) days in order of ascending dose levels.

One female in the control group (no. 54), one in the low dose group (no. 58) and two in the mid dose group (nos. 71 and 77) were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 90.9%, 90.9%, 81.8% and 100.0% in order of ascending dose level.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.4, 21.7, 21.2 and 21.5 days, in order of ascending dose levels.

CORPORA LUTEA COUNT

No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 14.0, 14.4, 13.3 and 14.1 in order of ascending dose levels.

IMPLANTATION RATE AND POST IMPLANTATION LOSS

No effects on implantation rate and post-implantation loss were noted.

The overall number of implantations per dam was 12.6, 13.1, 12.0 and 13.0 in order of ascending dose level. The overall mean number of post-implantation loss per dam was 0.7, 0.7, 0.8 and 0.9, respectively at the dose level of 0, 100, 300 and 1000 mg/kg body weight/day.

LITTER SIZE AT FIRST LITTER CHECK

There was no effect on mean value of living pups per dam at first litter check. Birth index was unaffected by treatment with the test item.

The overall mean numbers of living pups per dam at first litter check were 11.9, 12.4, 11.4 and 12.1, whereas birth indices (number of pups borne alive as a percentage of implantations) were 94.4%, 94.7%, 95.4% and 93.0% at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day, respectively.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

No changes in organ weights considered to be test item-related were noted at any dose level.

MACROSCOPICAL FINDINGS

- Males: There were no macroscopical findings noted at termination.

- Females: No test item-related findings were noted during necropsy of females at any dose level. Discoloration of ovaries was recorded for two females in the 100 mg/kg body weight/day dose group and for one female in the 300 mg/kg body weight/day dose group. Due to the lack of dose dependency and correlating histopathological findings this was considered to be incidental.

HISTOPATHOLOGY FINDINGS

There were no microscopic findings of the reproductive organs examined that could be attributed to treatment with the test item

Sperm staging: No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

All findings recorded such as focal tubular athrophy or tubular vacuolation in the testes, mononuclear infiltration in the epididymides, inflammation in the prostate gland were considered to be within the range of normal background alterations.

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

100 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Based on the reduction in food consumption and body weight as well as body weight gain in males treated at 300 and 1000 mg/kg bw/day

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

female

Basis for effect level:

other: No test-item related effects in females at 1000 mg/kg, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

for fertility

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum at 1000 mg/kg bw/day, the highest dose tested.

Remarks on result:

other: Generation not specified (migrated information)

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

Histopathological findings:

not examined

POSTNATAL LOSS ON DAYS 0 – 4 POST PARTUM

There was no effect on postnatal loss at any dose level.

One female pup was missing in litter 60 in group 2 and one female and one male pups each were missing in litters 73 and 74 in group 3, respectively on day 2 post partum.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related observations were noted in pups during the first litter check or during early lactation at any dose level.

A wound on the left hindpaw was noted for one male pup in a control litter (no. 47) and haematoma on the head was observed in one male pup (litter no. 60) in group 2.

Furthermore, no milk in stomach was noted for one female pup in group 2. A wound on the nose was found in one male pup in group 3 and absent tail was noted for one male pup in group 4.

As of their isolated occurrences these findings were considered to be incidental.

A wound on the left hindpaw of one control pup and on the nose in one mid dose group pup was recorded. Furthermore one pup had haematoma on the head in group 2 and another pup had missing tail in group 4. These findings were incidental and within the historical control data range.

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 40%, 50%, 59% and 49%, in order of ascending dose level. The statistical difference in sex ratio in group 3 was considered to be incidental due to the lack of dose dependency.

BODY WEIGHTS TO DAY 4 POST PARTUM

No effects on pup body weights were noted at any dose level.

Mean body weights of pups on day 1 post partum were: 6.2 g, 6.0 g, 5.9 g and 5.9 g, at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day respectively. Mean differences in body weights of pups during the first four days of the lactation period were +46.6%, +45.5%, +48.7% and +45.0%, respectively.

MACROSCOPICAL FINDINGS

No test item-related findings were found in pups at any dose level.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litter

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated (A)	11	11	11	11
Number of pregnant females	10	10	9	11
Numbers of non-pregnant females (B)	1	1	2	0
Number of females which reared their pups until day 4 post partum	10	10	9	11

(A)          Female no. 51 in the controls had no mating detected but was pregnant

(B)          Female nos. 54, 58, 71 and 77 were not pregnant

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Acid Brown 425 to rats. Acid Brown 425 was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Acid Brown 425 was administered to male rats for 28 days (except for one male up to 34 days) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (between 40 and 53 days for non-pregnant females and between 41 and 54 for dams).

Black colored feces seen in all test item-treated animals were considered to be a result of the staining properties of the dark brown test item.

Treatment with the test item at the high-dose level caused a reduction in food consumption, body weights and body weight gains in males mainly observed during the pre-pairing. There was a test item-related effect seen on body weights and body weight gains of males in the 300 mg/kg body weight/day dose group as well.

There were no test item-related effects found in any females in any dose group.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the food consumption, body weight and/or body weight gain effects in males at 1000 and/or 300 mg/kg body weight/day, the NOEL for systemic toxicity for males was considered to be 100 mg/kg body weight/day and 1000 mg/kg body weight/day for females. Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Acid Brown 425 on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

Four groups of 11 males and 11 females were treated by gavage with Acid Brown 425 once daily. Males were treated over a 14-day period following completion of pre-pairing period. Females were treated throughout gestation period up to the day 4 post partum.

 

The following dose levels were used:

 

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3:300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with highly purified water.

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

No test item-related clinical signs were noted at any dose level.

 

Black discoloration of feces was recorded in all males and females treated with the test item.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

At 1000 mg/kg body weight/day, mean food consumption of males was statistically significantly decreased during the pre-pairing period and this was considered to be a test item-related effect. There were no effects on food consumption of females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

At 1000 and at 300 mg/kg body weight/day the mean body weights and mean body weight gains of males were statistically significantly reduced during the pre-pairing period and the body weights in the pairing period as well. These were considered to be treatment-related effects. No effects on body weight or body weight gains were observed in females in any dose group.

 

At the dose level of 100 mg/kg body weight/day, body weights and body weight gain were not affected by the treatment.

 

REPRODUCTION AND BREEDING DATA

 

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

 

ORGAN WEIGHTS OF PARENTAL MALES

 

No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGY EXAMINATION OF PARENTAL ANIMALS

 

No findings related to the treatment with the test item were noted during macroscopical and histopathological examinations of reproductive organs in males or females at any dose level.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups at any dose level.

 

Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No effects on pup body weights or body weight gain were noted at any dose level.

 

MACROSCPICAL FINDINGS IN PUPS

 

No test item-related findings were found in pups at any dose level.

 

CONCLUSION

 

Based on the food consumption, body weight and/or body weight gain effects in males at 1000 and/or 300 mg/kg body weight/day, the NOEL for systemic toxicity for males was considered to be 100 mg/kg body weight/day and 1000 mg/kg body weight/day for females. The NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.

 

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:

2 (valid with restriction)
Screening study according to OECD guideline 421

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No effects on reproductive performance of rats at all were detected. The NOAEL was determined as 1000 mg/kg bw after treatment of paternal animals for at least 2 weeks and maternal animals for at least 2 weeks before mating and during mating and all of gestation until day 4 of lactation (ca. 7 weeks in total).

Short description of key information:
A oral screening assay according to OECD guideline 421 did not give any indication of effects on fertility or development of offspring in rats. Only slight effects were recorded in parental animals at the highest dose (1000 mg/kg bw).

Justification for selection of Effect on fertility via oral route:
recent guideline study under GLP

Effects on developmental toxicity

Description of key information

A oral screening assay according to OECD guideline 421 did not give ay indication of effects on fertility or development of offspring in rats. Only slight effects were recorded in parental animals at the highest dose (1000 mg/kg bw).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-May-2012 to 26-Nov-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well planned GLP and OECD guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 295 to 329 g (males) and 178 to 213 g (females)
- Identification: Cage card and individual animal number (ear tattoo).Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values for both temperature and humidity were outside these ranges but were transient variations and were considered not to have any influence on the study and, therefore, these data are not reported but retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) (batch/lot nos. 02105120601 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Route of administration:

oral: gavage

Vehicle:

other: Highly purified water (ELGA labwater)

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Acid Brown 425 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were divided into daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored in refrigerator (5 ± 3 °C) in brown bottles in daily aliquots.

Based upon the results of stability analyses performed within the Harlan Laboratories non-GLP study D51147 (Dose Range-Finding Study for a Reproduction/Developmental Toxicity Screen¬ing Test in the Han Wistar Rat), dose formulations were stable for at least one week in refrigerator.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (group 1, control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D51147, using dose levels of 0, 100, 300 and 1000 mg/kg/ day. No adverse toxic effects were found at any dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (group 1), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the responsible person for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS following an analytical procedure developed at Harlan Laboratories study D51160. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

RESULTS

The application formulations investigated during the study were found to comprise Acid Brown 425 in the range of 96.4% to 113.1% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Acid Brown 425 in the preparations was approved because single results found did not deviate more than 1.1% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature or eight days refrigerated due to recoveries which met the variation limit of ±10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Acid Brown 425 and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Duration of treatment / exposure:

Minimum 4 weeks (males) or approximately 7 weeks (females)

Frequency of treatment:

Once daily

Duration of test:

MALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days of treatment

FEMALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Necropsy: On day 5 post partum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of Acid Brown 425 on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and lactation periods (females) including the day before scheduled necropsy.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Maternal examinations:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY

Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology peer review was performed and the results included in the histopathology phase report.

Ovaries and uterine content:

The ovaries and the uterus were examined after termination on day 5 post partum. Examinations included the number of corpora lutea and the number of implantation sites.

Fetal examinations:

Not performed

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1. IN-LIFE DATA

VIABILITY/MORTALITY

All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS

Black colored feces were recorded in females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item.

In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in females at any dose level.

FOOD CONSUMPTION

There were no effects on food consumption of females at any dose level.

BODY WEIGHTS AND BODY WEIGHT GAIN

No test item-related effects on body weights and body weight gain of females were observed at any dose level.

2. TERMINAL FINDINGS

MACROSCOPICAL FINDINGS

No test item-related findings were noted during necropsy of females at any dose level. Discoloration of ovaries was recorded for two females in the 100 mg/kg body weight/day dose group and for one female in the 300 mg/kg body weight/day dose group. Due to the lack of dose dependency and correlating histopathological findings this was considered to be incidental.

HISTOPATHOLOGY FINDINGS

There were no microscopic findings of the reproductive organs examined that could be attributed to treatment with the test item

All findings recorded were considered to be within the range of normal background alterations.

Dose descriptor:

NOEL

Remarks:

for systemic toxicity in males

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOEL

Remarks:

for systemic toxicity in females

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOEL

Remarks:

for developmental toxicity

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: effect type not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Details on embryotoxic / teratogenic effects:
Not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

1. REPRODUCTION, BREEDING AND PUP DATA

 

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litter

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated (A)	11	11	11	11
Number of pregnant females	10	10	9	11
Numbers of non-pregnant females (B)	1	1	2	0
Number of females which reared their pups until day 4 post partum	10	10	9	11

(A) Female no. 51 in the controls had no mating detected but was pregnant

(B) Female nos. 54, 58, 71 and 77 were not pregnant

 

MATING PERFORMANCE AND FERTILITY

 

No effect on mating performance or fertility was observed at any dose level.

 

Mating of all test item-treated females was recorded during the first pairing period. No mating was detected for one control female (no. 51) but it was pregnant and delivered pups. The precoital time was not affected by the treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 5.0 (3), 4.1 (3), 2.4 (3) and 4.4 (3) days in order of ascending dose levels.

 

One female in the control group (no. 54), one in the low dose group (no. 58) and two in the mid dose group (nos. 71 and 77) were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 90.9%, 90.9%, 81.8% and 100.0% in order of ascending dose level.

 

DURATION OF GESTATION

 

No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.4, 21.7, 21.2 and 21.5 days, in order of ascending dose levels.

 

CORPORA LUTEA COUNT

 

No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 14.0, 14.4, 13.3 and 14.1 in order of ascending dose levels.

 

IMPLANTATION RATE AND POST IMPLANTATION LOSS

 

No effects on implantation rate and post-implantation loss were noted.

 

The overall number of implantations per dam was 12.6, 13.1, 12.0 and 13.0 in order of ascending dose level. The overall mean number of post-implantation loss per dam was 0.7, 0.7, 0.8 and 0.9, respectively at the dose level of 0, 100, 300 and 1000 mg/kg body weight/day.

 

LITTER SIZE AT FIRST LITTER CHECK

 

There was no effect on mean value of living pups per dam at first litter check. Birth index was unaffected by treatment with the test item.

 

The overall mean numbers of living pups per dam at first litter check were 11.9, 12.4, 11.4 and 12.1, whereas birth indices (number of pups borne alive as a percentage of implantations) were 94.4%, 94.7%, 95.4% and 93.0% at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day, respectively.

 

POSTNATAL LOSS ON DAYS 0 – 4 POST PARTUM

 

There was no effect on postnatal loss at any dose level.

 

One female pup was missing in litter 60 in group 2 and one female and one male pups each were missing in litters 73 and 74 in group 3, respectively on day 2 post partum.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related observations were noted in pups during the first litter check or during early lactation at any dose level.

 

A wound on the left hindpaw was noted for one male pup in a control litter (no. 47) and haematoma on the head was observed in one male pup (litter no. 60) in group 2.

 

Furthermore, no milk in stomach was noted for one female pup in group 2. A wound on the nose was found in one male pup in group 3 and absent tail was noted for one male pup in group 4.

 

As of their isolated occurrences these findings were considered to be incidental.

 

A wound on the left hindpaw of one control pup and on the nose in one mid dose group pup was recorded. Furthermore one pup had haematoma on the head in group 2 and another pup had missing tail in group 4. These findings were incidental and within the historical control data range.

 

PUP SEX RATIOS

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

At first litter check, percentages of male pups were 40%, 50%, 59% and 49%, in order of ascending dose level. The statistical difference in sex ratio in group 3 was considered to be incidental due to the lack of dose dependency.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

No effects on pup body weights were noted at any dose level.

 

Mean body weights of pups on day 1 post partum were: 6.2 g, 6.0 g, 5.9 g and 5.9 g, at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day respectively. Mean differences in body weights of pups during the first four days of the lactation period were +46.6%, +45.5%, +48.7% and +45.0%, respectively.

 

MACROSCOPICAL FINDINGS OF PUPS

 

No test item-related findings were found in pups at any dose level.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY/MORTALITY

 

All animals survived the scheduled study period.

 

CLINICAL SIGNS OR OBSERVATIONS

 

Black colored feces were recorded in males of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item.

 

In one male (no. 35) in the high dose group a wound in the neck region was recorded for a few days in the pairing period. No other clinical signs were noted in males at any dose level.

 

FOOD CONSUMPTION

 

At 1000 mg/kg body weight/day, mean food consumption of males was statistically significantly decreased between days 1 - 14 in the pre-pairing period. This was considered to be a test item-related effect.

 

No effect on food consumption of males was observed in the 300 and 100 mg/kg body weight/day dose groups.

 

BODY WEIGHTS AND BODY WEIGHT GAIN

 

At 1000 and at 300 mg/kg body weight/day, statistically significantly reduced body weights were noted in males from day 11 until day 14 of the pre-pairing period and throughout the pairing period.

 

At 1000 and 300 mg/kg body weight/day, the mean body weight gains of males were statistically significantly reduced from early pre-pairing period until the end of this period. At 300 mg/kg body weight/day, the body weight gain was statistically significantly lower on certain days during the pairing period too (on Days 4-8 and 11, 12) but without toxicological relevance. The body weight gains were statistically significantly reduced and showed a dose dependent pattern in the pre-pairing period beginning from Day 2 and Day 3 of treatment in groups 4 and 3, respectively. The values were lower than in controls and in the low dose group, therefore the reduced body weight gains in the pre-pairing period were considered to be test item-related effects in groups 3 and 4.

 

The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day were respectively: +16%, +15%, +12% and +10% during the pre-pairing period (percentages refer to the body weight gain within the period).

 

3. TERMINAL FINDINGS IN PARENTAL MALES

 

ORGAN WEIGHTS

 

No changes in organ weights considered to be test item-related were noted at any dose level.

 

MACROSCOPICAL FINDINGS

 

There were no macroscopical findings noted at termination.

 

HISTOPATHOLOGY FINDINGS

 

There were no microscopic findings of the reproductive organs examined that could be attributed to treatment with the test item

 

Sperm staging: No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

 

All findings recorded such as focal tubular athrophy or tubular vacuolation in the testes, mononuclear infiltration in the epididymides, inflammation in the prostate gland were considered to be within the range of normal background alterations.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Acid Brown 425 to rats. Acid Brown 425 was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Acid Brown 425 was administered to male rats for 28 days (except for one male up to 34 days) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (between 40 and 53 days for non-pregnant females and between 41 and 54 for dams).

Black colored feces seen in all test item-treated animals were considered to be a result of the staining properties of the dark brown test item.

Treatment with the test item at the high-dose level caused a reduction in food consumption, body weights and body weight gains in males mainly observed during the pre-pairing. There was a test item-related effect seen on body weights and body weight gains of males in the 300 mg/kg body weight/day dose group as well.

There were no test item-related effects found in any females in any dose group.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the food consumption, body weight and/or body weight gain effects in males at 1000 and/or 300 mg/kg body weight/day, the NOEL for systemic toxicity for males was considered to be 100 mg/kg body weight/day and 1000 mg/kg body weight/day for females. Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Acid Brown 425 on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

Four groups of 11 males and 11 females were treated by gavage with Acid Brown 425 once daily. Males were treated over a 14-day period following completion of pre-pairing period. Females were treated throughout gestation period up to the day 4 post partum.

 

The following dose levels were used:

 

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3:300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with highly purified water.

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

No test item-related clinical signs were noted at any dose level.

 

Black discoloration of feces was recorded in all males and females treated with the test item.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

At 1000 mg/kg body weight/day, mean food consumption of males was statistically significantly decreased during the pre-pairing period and this was considered to be a test item-related effect. There were no effects on food consumption of females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

At 1000 and at 300 mg/kg body weight/day the mean body weights and mean body weight gains of males were statistically significantly reduced during the pre-pairing period and the body weights in the pairing period as well. These were considered to be treatment-related effects. No effects on body weight or body weight gains were observed in females in any dose group.

 

At the dose level of 100 mg/kg body weight/day, body weights and body weight gain were not affected by the treatment.

 

REPRODUCTION AND BREEDING DATA

 

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

 

ORGAN WEIGHTS OF PARENTAL MALES

 

No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGY EXAMINATION OF PARENTAL ANIMALS

 

No findings related to the treatment with the test item were noted during macroscopical and histopathological examinations of reproductive organs in males or females at any dose level.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups at any dose level.

 

Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No effects on pup body weights or body weight gain were noted at any dose level.

 

MACROSCPICAL FINDINGS IN PUPS

 

No test item-related findings were found in pups at any dose level.

 

CONCLUSION

 

Based on the food consumption, body weight and/or body weight gain effects in males at 1000 and/or 300 mg/kg body weight/day, the NOEL for systemic toxicity for males was considered to be 100 mg/kg body weight/day and 1000 mg/kg body weight/day for females. The NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/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:

2 (valid with restriction)
Screening study according to OECD guideline 421

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

No effects on litter parameters or offspring of rats at all were detected. All pups developed normally. The NOAEL was determined as 1000 mg/kg bw after treatment of paternal animals for at least 2 weeks and maternal animals for at least 2 weeks before and during all of gestation until day 4 of lactation.

Justification for selection of Effect on developmental toxicity: via oral route:
recent guideline study under GLP

Justification for classification or non-classification

not classified;

The NOAEL for reproductive effects was at least 1000 mg/kg bw.