Reaction mass of sodium(E)-6,7'-carbonylbis(azanediyl)bis(4-hydroxy-3-((E)-phenyldiazenyl) naphthalene-2-sulfonate) and disodium 3-[(4-acetamidophenyl)azo]-4-hydroxy-7-[[[[5-hydroxy-6-(phenylazo)-7-sulphonato-2-naphthyl]amino]carbonyl]amino]naphthalene-2-sulphonate and disodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-(phenylazo)naphthalene-2-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL > 1000 mg/Kg P

NOAEL > 1000 mg/Kg F1

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 11 wks
- Weight at study initiation: Males 322-375 g, Females 205-238 g
- Housing: individual (after mating)
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet ad libitum
- Water: tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The dose formulations were be prepared fresh daily using the test item as supplied by the Sponsor.
Direct Red 239 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days at maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; this day was referred to as day 0 post coitum
- After successful mating each pregnant female was caged (how): individual

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group (middle only) 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. During week 3 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 toHarlan Laboratories Ltd and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

once every day

Details on study schedule:

Males and Females were treated with the test substance for 14 days prior to mating (10 ml/kg bw); after 14 days, one male was paired with one female and animals treated for 14 days at maximum; males and females were then seperated; males were subjected to necroscopy, whereas treatment of females continued until day 3 post partum
Males were sacrificed after they had been treated for at least 28 days. Dams and pups were sacrificed on day 4 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg/d
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available toxicological data of the test compound and of a closely related compound

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were examined macroscopically.

Postmortem examinations (parental animals):

All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.
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.
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. The ovaries (including oviduct) and uterus (including cervix and vagina) from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. Slides of testes, epididymides and ovaries from all animals of the control and high-dose groups were examined by the study pathologist. 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.

Postmortem examinations (offspring):

Dead pups were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, organ 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:

Fertility index [%] = (Number of pregnenat females / Number of females paired) x 100
Conception index [%] = (Number of pregnenat females / Number of females mated) x 100
Gestation index [%] = (Number of femeales bearing live pups/ Number of pregnant females) x 100

Offspring viability indices:

Viability index [%] = (Number of live pups on Day 4 post-partum / Number of pups born alive) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived the scheduled study period.
Red colored feces were recorded in males and females of all groups treated with the test item from the start of dosing until necropsy. Severity increased with increasing dose levels. No clinical signs were noted in males or females at any dose level.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no effects on food consumption at any dose level in males and females.
The overall differences in mean body weight gain for males at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were : +12%, +12%, +11% and +9% during the pre-pairing period and +3%, +3%, +2% and +2% during the pairing period (percentages refer to the body weight gain within the period); The overall differences in mean body weight gain for females at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were : +6%, +7%, +5% and +4% during the pre-pairing period, +58%, +54%, +53% and +56% during the gestation period and +9%, +6%, +6% and +5% during the lactation period (percentages refer to the body weight gain within the period).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effect on mating performance or fertility was observed at any dose level. Mean (median) precoital times calculated for the first pairing period were 3.4 (3), 3.6 (3), 4.0 (3) and 4.5 (3) days in order of ascending dose levels.
Two females in the control group (nos. 52 and 55), two of the low dose group (nos. 59 and 65), one of the mid dose group (no. 70) and three of the high dose group (nos. 78, 81 and 87) were not pregnant -> Fertility Index = Conception Index = 81.8%, 81.8%, 90.9% and 72.7% in order of ascending dose levels.
No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 83). At termination on day 25 post coitum, only implantation sites were found in this female -> gestation index = 100, 100, 100, 87.5 % in order of ascending dose levels.
None of these parameters were statistically significantly different compared to the control group and in the range of the historical control data and therefore considered to be a result of biological variability.
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.7, 21.8, 21.8 and 21.9 days, in order of ascending dose levels.
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 17.0, 17.6, 15.8 and 15.7 in order of ascending dose levels.
No effects on implantation rate and post-implantation loss were noted. An increase in the total number of post-implantation loss was noted at 300 mg/kg bw/day but due to the absence of any dosage relationship this was considered to be incidental. The overall number of implantations per dam was 14.4, 12.7, 12.6 and 12.7 in order of ascending dose level. The overall mean number of post-implantation loss per dam was 0.7, 1.1, 1.5 and 1.0 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No changes in organ weights considered to be test item-related were noted at any dose level

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no macroscopical findings that were considered to be related to treatment with the test item. All findings occurred in individual males or females and were considered to be within the range of the normal background alterations.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopic findings 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:

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 effects observed

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

VIABILITY (OFFSPRING)
The overall mean numbers of living pups per dam at first litter check were 13.8, 11.6, 11.1 and 11.7, whereas birth indices (number of pups borne alive as a percentage of implantations) were 95.4%, 91.2%, 88.1% and 92.1% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. There was no effect on postnatal loss at any dose level. At the dose level of 300 mg/kg bw/day, one female (no. 67) gave birth to two dead pups. Four pups of this dam died within the first four days after delivery. This isolated occurrence was considered to be incidental.

CLINICAL SIGNS (OFFSPRING)
No observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by exposure to the test item at any dose level.

BODY WEIGHT (OFFSPRING)
No effects on pup body weights were noted at any dose level.
Mean body weights of pups on day 1 post partum were: 5.8 g, 6.3 g, 6.0 g and 6.2 g, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively, body weight gain of pups during the first four days of the lactation period was +45.4%, +47.9%, +54.7% and +43.0%, respectively.

GROSS PATHOLOGY (OFFSPRING)
No findings were found in pups at any dose level.

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 adverse effects observed

Reproductive effects observed:

not specified

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	11	11	11	11
Number of pregnant females (A)	9	9	10	8
Numbers of females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	9	9	10	7

(A)   Female nos. 52, 55, 59, 65, 70, 78, 81 and 87 were not pregnant

(B)   Female no 83 had only implantation sites

Conclusions:

NOAEL > 1000 mg/Kg P
NOAEL > 1000 mg/Kg F1

Executive summary:

Substance was tested according to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) on wistar rats by oral gavage, at dose 100, 300, 1000 mg/kg/d, during minimum 4 weeks for males and approximately 7 weeks for females.

Results

No adverse effects observed at dosage of 1000 mg/kg

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the screening test for reproductive toxicity conducted according to the OECD guideline 421 no adverse effects were reported also at the highest dose tested of 1000 mg/kg (BASF, 2013).

No clinical signs were noted in males or females at any dose level. No effect on mating performance or fertility, on duration of gestation, on corpora lutea count were observed at any dose level and no effects on implantation rate and post-implantation loss were noted. Furthermore no changes in organ weights, no macroscopical and no microscopic findings were observed that were considered to be related to treatment with the test item.

Considering offsprings the overall mean numbers of living pups per dam at first litter check were 95.4%, 91.2%, 88.1% and 92.1% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. There was no effect on postnatal loss at any dose level.

No observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by exposure to the test item at any dose level. No effects and no findings on pups were noted at any dose level.

Short description of key information:

NOAEL > 1000 mg/Kg P

NOAEL > 1000 mg/Kg F1

Effects on developmental toxicity

Description of key information

NA

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Description of key information

NOAEL > 1000 mg/Kg P

NOAEL > 1000 mg/Kg F1

Additional information

In the reproduction/developmental toxicity screening test were not observed adverse effects on fertility and results are reasonably clear to exclude the suspect of any developmental toxicant effects; therefore there are no concerns to consider further investigations.