REAKTIV-GELB F-68 072 FW

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reactive Yellow F68072 induced polyuria and polydipsia at 750 mg/kg/day dose level on parental generation of CRL:(WI)BR rats during the course of a one generation reproduction toxicity study. This is a result of the hight lithium content in the test material, as polyuria and polydipsia are common side effects of lithium.

Reproductive performance of males and females were unaffected by the treatment. The mortality of pups was a slightly higher at 750 mg/kg bw/day,  as a consequence of maternal lithium effects (polyuria). There was no effect on postnatal development of viable pups.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2006 to 10 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

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
- Source: TOXI COOP Ltd. 1032 Budapest, Cserkesz u. 90.
- Age at study initiation: Males: less than 9 weeks old
Females: at least 9 weeks old
- Housing: Before mating: 4 male animals/ cage
5 female animals/ cage
Mating period: male animals: individual caging,
Mating hours: 1 male and 1 female / cage
During pregnancy: in groups of 1 to 4 animals
Delivery and nursing: individual caging
- Diet (ad libitum): sniff SM R/M-Z+H autoclavable complete feed for rats and mice - breeding and maintenance
- Water (ad libitum): tap
- Acclimation period: 29 days for male rats
6 days for female rats

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 June 2006 To: 10 November 2006

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in distilled water in concentrations of 10 mg/mL, 30 mg/mL and 75 mg/mL. The pH of dosing solutions were adjusted to pH=7.0 with 10 M NaOH (solid NaOH: Batch No.: KBM 5080902, Expiry date: November 2009) during the first three weeks treatment. The results of the preliminary study (two days treatment) regarding the pH influence on digestive system were not supported by the observations after two weeks treatment therefore pH was not adjusted after three weeks treatment. Formulations were prepared daily except weekends. Dosing solutions were stable for at least 24 hours at room temperature and 4 days refrigerated. The stock solution was stable for at least 10 days at 2 to 8°C. Homogeneity of test item in this vehicle was analytically proven. Analytical control of dosing solutions was performed five times during the treatment period on Days 0, 56, 77, 99, and 131.

VEHICLE: Distilled water
- Concentration in vehicle: 10 mg/mL, 30 mg/mL and 75 mg/mL
- Amount of vehicle: 10 mL/kg body weight
- Lot/batch nos.: 0503-0306; 85 10604; 3490306; 3530306; 3630306; 53 10506; 6650706

Details on mating procedure:

Mating of the animals began approximately 11 weeks after starting of the treatment of male animals and 4 weeks after starting of the treatment of female animals.

One female was placed to the male of the same dose group (1:1 mating) until copulation occurred or two weeks had elapsed. Mating time was about 3 hours a day in the morning. Thereafter each morning vaginal smear was prepared and stained with 1 % aqueous methylene blue solution.

The day of mating (presence of vaginal plug or sperms in the vaginal smear) was considered as day 0 of pregnancy (as defined by OECD's TG 415). Sperm positive females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions was performed five times during the treatment period on Days 0, 56, 77, 99, and 131 with a validated HPLC method.

Duration of treatment / exposure:

Males: pre-mating: 81 days
pairing: 12 days
post-mating: 4 to 5 days
Females: pre-mating: 27 days
pairing: 12 days
gestation: 22 to 24 days
lactation: 21 to 23 days

Frequency of treatment:

daily

Details on study schedule:

Pre-treatment period
Animal arriving: Male (M): 31 May 2006; Female (F): 16 August 2006
Veterinary control, acclimatisation: M: 31 May - 28 June 2006 (29 days); F: 16 - 21 August 2006 (6 days)
Animal identification/body weight measurement/randomisation: M: 28 June 2006; F: 21 August 2006

Treatment period
Pre-mating period: M: 29 June - 17 September 2006 (81 days); F: 22 August - 17 September 2006 (27 days)
Examination of estrous cycle: 22 August - 29 September 2006
Pairing period: 18 - 29 September 2006 (12 days)
Mating period: 18 - 27 September 2006 (10 days)
Gestation periods: 18 September - 19 October 2006 (32 days)
Lactation periods: 10 October - 10 November 2006 (32 days)

Clinical observation: M: Daily from 29 June 2006 - up to the necropsy; F: Daily from 22 August 2006 - up to the necropsy
Body weight measurement: M: 29 June 2006, then weekly
F: 22 August 2006, then weekly prior to and during the mating period,
- On gestational days 0, 4, 7, 10, 14, 17 and 21
- On postpartal days 0, 4, 7, 10, 14, 17 and 21
Food consumption measurement: M: 06 July 2006, then weekly prior to the mating
F: 29 August 2006, then weekly prior to the mating
- On gestational days 0, 7, 14 and 21
- On postpartal days 0, 4, 7, 14 and 21

PUPS
Body weight measurement: 10 October - 09 November 2006
- On postnatal days 0, 4, 7, 14 and 21
Surface righting reflex: 10 - 20 October 2006 - At the birthday
Pinna detachment: 12 - 22 October - On postnatal day 2
Eye opening: 24 October - 03 November 2006 - On postnatal day 14

Termination
Necropsy: M: 03, 04 October 2006
Dead animals: No.: 408: 28 September 2006
No.: 418: 05 August 2006
Dams delivered: 31 October - 10 November 2006
Dams not delivered: 25 October 2006
Females not mated: 04 October 2006
Offspring: sacrificed on postnatal day 21, 22 or 23

The end of the in life phase: 10 November 2006

Dose / conc.:

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

Remarks:

10 mg/ml analytical conc.

Dose / conc.:

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

Remarks:

30 mg/ml analytical conc.

Dose / conc.:

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

Remarks:

75 mg/ml analytical conc.

No. of animals per sex per dose:

24 males/group
25 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The doses were chosen on the basis of the results of previous rodent studies and a preliminary toxicity study with the test item in rats. A group of male animals was treated at 1000 mg/kg bw/day dose for about 2 weeks before mating. During this period, a decision was made to eliminate this dose level from main part of the study before the female treatment has been started because of deaths of several male rats (9/24) treated with 1000 mg/kg bw/day.

Parental animals: Observations and examinations:

PARENTAL (P) GENERATION

Clinical observations

A general clinical observation was made once a day, after treatment at approximately the same time. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachryrnation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

Morbidity and mortality
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). Animals found dead were subjected to gross pathology and all organs were preserved in 10 % buffered formalin solutions but testes were preserved in Bouin's solution.

Body weight
All parent animals were weighed with accuracy of 1 g.
Parent (P) male animals were weighed and evaluated weekly (on the same day) prior to and during the mating period.
Parent females were weighed and evaluated weekly (on the same day) prior to and during the mating, on gestation days 0, 7, 14 and 21 and on postpartal days 0, 7, 14 and 21. Body weight of the female animals was weighed on gestation and postpartal days 4, 10 and 17 in order to make sure the accurate treatment, but these data were not evaluated statistically.

Food consumption
Food consumption was determined weekly (on the same day) by reweighing the nonconsumed diet with accuracy of 1 g during the pre-mating period and during the gestation period. After parturition, the non-consumed diet was weighed on the same days as pups:
- On gestational days 0, 7, 14 and 2 1
- On postpartal days 0, 4, 7, 14 and 2 1

Oestrous cyclicity (parental animals):

Examination of the oestrous cycle
Vaginal smear of all the females was prepared daily during the pre-mating period four weeks before the mating period and during the mating period. The vaginal smears were stained with 1 % aqueous methylene blue solution and were examined with light microscope.

Sperm parameters (parental animals):

Not assessed

Litter observations:

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All changes were recorded.

Observation of the delivery process
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All changes were recorded.

Observation of the nursing instinct
Dams were observed whether they made a nest from the bedding material and cover their new-born pups or not. The efficiency of the lactation and nursing was observed by the presence of milk in the pups' stomach. All observations were recorded.


STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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, physical or behavioural abnormalities, development

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were subjected to necropsy following terminal anaesthesia 4 -5 days after the mating period
- Female animals:
- Non-mated female animals were subjected to necropsy following terminal anaesthesia 5 days after the mating period.
Occasional implantations in the uterus were checked. If there was implantation in the uterus, corpora lutea were counted, too
- Animals, which failed to deliver up to gestation day 24 were subjected to necropsy following terminal anaesthesia
- Dams with viable pups were subjected to necropsy following terminal anaesthesia after post-partal day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs of parental animals or representative samples thereof were preserved all in 4% neutral formaldehyde solution but testis in Bouin's solution for histological examination:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), larynx, femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, Harderian glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve, aorta.
Gross lesions, the ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles, prostate, coagulating gland and pituitary gland were subjected to histological examinations.
Histological examinations were conducted in all control and high dose treated animals. A full histological examination was performed for animals, which were found dead during the study.
Reproductive organs of animals suspected of fertility were subjected to microscopic examination in low and medium dose groups.

Postmortem examinations (offspring):

SACRIFICE
- All dead pups were dissected to find the cause of the death. It was determined whether the dead newborn was live-born or stillborn by lung flotation test.
- The F1 offspring were sacrificed without necropsy following terminal anaesthesia on postnatal day 2 1,22 or 23.

Statistics:

STATISTICAL EVALUATION
The following parameters were evaluated:

PARENTAL MALES (P)
- Clinical observations
- Body weight (g)
- Body weight gain (g)
- Food consumption (g)
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Fertility index (%)
- Necropsy findings (%)

PARENTAL FEMALES (P)
- Clinical observations
- Body weight (g)
- Body weight gain (g)
- Food consumption (g)
- Number of not mated females
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Copulatory index (%)
- Fertility index (%)
- Gestation index (%)
- Number of oestrous periods (until mating)
- Oestrous length (day, until mating)
- Duration of pregnancy (day)
- Implantations 1 dams
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)
- Necropsy findings (%)
- Histopathological findings (%)

OFFSPRING (F1)
- Mean body weight per litter on postnatal days 0,4,7, 14 and 21
- Mean body weight gain per litter between postnatal days 0-4,4-7, 7-14, 14-21 and
0-2 1
- Number of live births per litter, and number of viable pups per litter on postnatal
days 0,7, 14 and 21
- Extra uterine mortality of pups on postnatal days 0,2 1
- Viability index (%)
- Lactation index (96)
- Sex ratio % (on postnatal days 0 and 21)
- Surface righting reflex (%)
- Pinna detachment (%)
- Eye-opening (%)
- Necropsy findings (%)


The statistical evaluation of appropriate data was done SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. At significant result at Bartlett's test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann- Whitney U-test. The Chi² test was performed if feasible.

Reproductive indices:

Copulatory index (%): Number of sperm positive females x 100 / Number of mated females
Fertility index - Males (%): Number of fertile males x 100 / Number of mated males
Fertility index - Females (%): Number of pregnant females x 100 / Number of mated females
Gestation index (%): Number of females with viable pups x 100 / Number of pregnant females
Sex ratio: (Number of pups examined - Number of males (females)) x 100 / Number of pups examined

Offspring viability indices:

Intra uterine mortality: (Number of implantations - Number of newborns) x 100 / Number of implantations
Total mortality: (Number of implantations - Number of viable pups) x 100 / Number of newborns
Viability index (%): Number of viable pups on day 4 (7, 14, 21) x 100 / Number of viable pups on day 0 (4, 7, 14)
Lactation index (%): Number of viable pups on day 21 x 100 / Number of viable pups on day 0 of lactation
Surface righting reflex, Pinna detachment, Eye-opening: (Number of pups examined - Number of pups with negative response) x 100 / Number of pups examined

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

An increased water consumption and urine excretion related to the test item was found at 750 mg bw/day group (male and female animals) from day 2 until the termination of the treatment. This is due to the high lithium salt content, as polyuria and polydidpsia are a common side-effect for lithium.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 750 mg/kg bw/day group, decreased activity and piloerection were noted for
animal No.: 408 on days 89 and 90 resulting in death of this animal next day.
One animal (No.: 418) was found in prone position with closed eyes and onvulsions
immediately after the treatment (day 37) and then was died. Histopathology reveled misdosing for both animals.
There were no clinical sings in female animals except for one pregnant rat (No.: 338) at 300 mg/kg bw/day. Paleness and piloerection were observed from gestational day 21 to gestational day 23 and 27, respectively. Enlarged spleen and early death was recorded at the gross pathology on gestation day 28. Histological examination revealed splenic hyperplasia as individual disorder.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SYMPTOMS AND MORTALITY
An increased water consumption and urine excretion related to the test item was found at 750 mg bw/day group (male and female animals) from day 2 until the termination of the treatment. There were no other test item related clinical signs at 100,300 or 750 mgkg bw/day doses.
Individual clinical observations noted for single male animals at all doses were as follows:
In the 100 mg/kg bw/day group, damage of the left eye was observed from day 70 up to the termination.
In the 300 mg/lg bw/day group, alopecia was found on the forelimbs from day 28 up to the necropsy.

Mortality
In the 750 mg/kg bw/day group, decreased activity and piloerection were noted for One animal on days 89 and 90 resulting in death of this animal next day. One animal was found in prone position with closed eyes and convulsions immediately after the treatment (day 37) and then was died.
There were no clinical signs in female animals except for one pregnant rat at 300 mg/kg bw/day. Paleness and piloerection were observed from gestational day 21 to gestational day 23 and 27, respectively. Enlarged spleen and early death was recorded at the gross pathology on gestation day 28. Histological examination revealed splenic hyperplasia as individual disorder.

BODY WEIGHT AND BODY WEIGHT GAIN
Pre-mating period.

100 mg/kg bw/day
In the male animals, the mean body weight gain slightly exceeded the control value on week 11.
In the female animals, the mean body weight gain was slightly higher on week 4.

300 mg/kg bw/day
In the male animals, the mean body weight gain was slightly above the mean control value on week 11.
In the female animals, the mean body weight gain was slightly higher than the control on week 4.

750 mg/kg bw/day
In the male animals, the mean body weight gain slightly exceeded the control value on weeks 2,3, and 9 and it was less on week 6.
In the female animals, the mean body weight gain was slightly exceeded the control value on week 2.

Gestation period
There were no differences in the body weight and body weight gain between the control and Reaktiv Gelb F68072 treated animals at any dose level.

Lactation period
300 mg/kg bw/day
A slightly less body weight gain was noted on the first week.

750 mg/kg bw/day
The mean body weight gain was slightly exceeded the control value on week 2. In summary: No test item influence on the body weight development was found. The body weight was comparable in the control and Reaktiv Gelb F68072 FW treated animals in the course of the study. There were no differences between the control and test item treated groups either during the gestation or lactation periods. The summarised body weight gain was similar in the control and test item treated groups during the pre-mating, gestation and lactation periods. Slight deviations from the control value occurred in some cases without any dose relevance and these were not considered to be test item related.

FOOD CONSUMPTION
Pre-mating period

100 mg/kg bw/day
There were no differences in the mean daily food intake between the control and Reaktiv Gelb F68072 FW treated animals (male and female).

300 mg/kg bw/day
In the male animals, the mean daily food consumption was slightly above the mean control value on week 8.
In the female animals, the mean daily food consumption exceeded the mean control value on week 3.

750 mg/kg bw/day
In the male animals, the mean daily food consumption was higher (8-12 %) than the mean control value during the entire pre-mating period except weeks 1 and 6. In the female animals, the mean daily food consumption was less on week 1 and was higher than the mean control value on weeks 3 and 4 of the pre-mating period.

Gestation period

100 mg/kg bw/day
There were no differences in the mean daily food intake between the control and Reaktiv Gelb F68072 FW treated animals.

300 mg/kg bw/day, 750 mg/kg bw/day
The mean food consumption was higher during the whole gestation period with statistical significance on week 2.

Lactation period
100 mg/kg bw/day
There were no differences in the mean daily food consumption between the control and Reaktiv Gelb F68072 FW treated animals.

300 mg/kg bw/day
The mean daily food intake was less than the control value on week 1.

750 mg/kg bw/day
The mean food consumption was slightly less than the control value on week 1 of the lactation period.

In summary: A slightly higher mean daily food consumption was noted for male and female animals at 750 mg/kg bw/day almost the entire pre-mating period and for female animals during the gestation period. Considering that there were no body weight differences, a test item influence on the efficacy of feeding or on the metabolism cannot be excluded. It should be mentioned however, the food consumption was not influenced during the lactation period.

OESTROUS CYCLE
There were no test item related differences in the oestrous cycle during the four weeks of the pre-treatment period. The number of animals with regular cycles, the number of animals with irregular cycles, the number of cycles, the number of days in prooestrous, the number of days in oestrous, the number of days in diestrous and number of animals in prolonged oestrous diestrous were considered to be normal in all groups.

DELIVERY DATA
There were no differences between the control and test item treated groups in percentage of dams delivered (calculated by the number of pregnant females), percentage of dams with viable foetuses, in live birth index and in lactation index. No test item effect were found either in the litter mean of the number of implantations, number of total births, number of liveborns and stillborns. No stillborns were found in the 100 mg/kg bw/day and 750 mg/kg bw/day groups.

The mean duration of pregnancy (22.55 days) was slightly longer in dams treated with 750 mg/kg bw/day dose of Reaktiv Gelb F68072 FW than in the control.

In summary: The gestation period was slightly prolonged at 750 mg/kg bw/lday: 10/22 dams delivered on day 23, one on day 24 in whereas the control group had 2/23 dams delivered on gestation day 23 all other on gestation day 22 or 21. However, this was not considered to be significant biologically, because it is specific for this species strain according to the laboratories Historical Control data (Gestation length: 22.17 +/-0.44 days, min: 2 1 days, Max 23 days, n= 41).

There were no test item related alterations in the delivery data of Reaktiv Gelb F68072
FW treated dams as compared to the control value.

REPRODUCTIVE PERFORMANCE
The number and percentage of mated and fertile male animals, the copulatory and fertility indices were not affected by the treatment. The number and percentage of fertile males and fertility index were the lowest in 100 mg/kg bw/day group. There were no differences between the control and test item treated groups in the number and percentage of sperm positive (mated) female animals and copulatory index. The number and percentage of non-pregnant females was the highest in the low dose group (100 mg/kg bw/day group). The percentage of pregnant animals was less than the control, consequently the fertility index was less in the 100 mg/kg bw/day group than that in the control group. The percentage of pregnants with liveborn was less than the control value and number of pregnants not delivered exceeded the control value in 300 mg/kg bw/day group, consecutively the gestation index was less in 300 mg/kg bw/day group than that in the control group. The mean precoital interval was similar in the experimental groups.

In summary: No test item effect was found on the reproductive ability of male and female animals. There were no significant differences between the control and Reaktiv Gelb F68072 FW treated groups. The above detailed slight deviations from control were observed in the low (100 mg/kg bw/day ) and middle (300 mg/kg bw/day ) groups but not in the high (750 mg/kg bw/day ) dose group and were considered to be accidental and independent from the treatment.


NECROPSY

Male animals

Control group
In the lungs sporadic pinhead-sized greyish white foci, pinprick-sized haemorrhages and in the kidneys unilateral pyelectasia (were observed.

100 mg/kg bw/day

The eye of single animal was traumatised and pinprick-sized haemorrhages in the lungs were noted for several rats . Pale and nutmeg-like patterned liver and unilateral renal pyelectasia were found in single animals.

300 mg/kg bw/day

Pinprick-sized haemorrhages were observed in the lungs.

750 mg/kg bw/day

In animals found dead, dark red lungs, yellow content in the stomach and empty intestines were observed. In the surviving animals, reddish mottled colour and pinprick-sized haemorrhages were noted in the lungs.

Female animals

Dams
Control

In the lungs pinprick-sized haemorrhages were noted for several dams. Pinprick-sized haemorrhages in the lungs and early embryonic death in the uterine horns were observed in animal No.: 150 (not delivered) on gestational day 31.

100 mg/kg bw/day

In the lungs pinprick-sized haemorrhages were noted.

300 mg/kg bw/day
In the lungs pinprick-sized haemorrhages were found . Enlarged spleen and early embryonic death in the uterine horn were observed in two dams,
which not delivered. Late embryonic death was noted for animal No.: 335.

750 mg/kg bw/day

In the lungs pinprick-sized haemorrhages and pale liver were observed.

Non-pregnant and not mated animals

Control

In the only non-pregnant control animal (No.: 136) pinprick-sized haemorrhages were found in the lungs.

100 mg/kg bw/day
In the non-pregnant animals, in the lungs pinprick-sized haemorrhages, in the not mated rat severe hydrometra were observed.

300 mg/kg bw/day

Pinprick-sized haemorrhages in the lungs were observed in the only non-pregnant animal.

In the not mated rat, pinprick-sized haemorrhages in the lungs and severe hydrometra in the uterus were noted.

750 mg/kg bw/day
No macroscopic findings were found in non-pregnant and not mated animals.

In summary: Gross necropsy revealed no test item related macroscopic findings. Pulmonary alterations of the dead animals are indicative of suffocation as cause of the death. In surviving animals, reddish mottled colour and pinprick-sized haemorrhages in the lungs were related to the exsanguination process. These commonly occur in untreated animals after exsanguination. Sporadic pinhead-sized greyish white foci in the lungs, pale and nutmeg-like pattern of liver, enlargement of the spleen, renal pyelectasia, trauma on the eye were considered as individual alterations commonly seen in untreated experimental and rats.

HISTOPATHOLOGY
Male animals
In the dead animals, (750 mg/kg bw/day , No.: 408 and 418) in the lungs diffuse congestion and alveolar oedema were related to suffocation as the cause of death. In animal No.: 408, subacute focal purulent-necrotic inflammation in the wall of oesophagus due to mechanical injury at the oral gavage and serous-purulent inflammation in the adventitia of thoracic aorta as an accompanying reaction of oesophageal lesion were observed. These lesions were independent of the test item.

Control group
In the lungs foamy cells and unilateral pyelectasia in the kidneys were observed.

100 mg/kg bw/day
One-side pyelectasia (in the kidney and zonal vacuolisation of hepatocytes were noted for single animals subjected to histological examinations on the basis of the results of macroscopic findings.

750 mg/kg bw/day
There were no histological lesions in male animals. The examined organs of reproductive system (testes, epididymides, seminal vesicles, prostate, coagulate gland) were considered to be normal histologically in all (control and treated) groups. The various spermatogenic cells, representing different phases in the development and differentiation of the spermatozoons were the same in quantity and morphologically in the testes and epididyrnides of animals belonging to the control and treated groups. The histological picture of seminal vesicles, prostate and coagulating gland was
normal in all cases.

Female animals

Dams

Control group,

In dams which delivered, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortex of ovaries in all female animals contained primordial, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. Purulent endometritis, necrotic debris and dilatation of uterus (1124) were observed in the dam not delivered (No.: 150).

300 mglkg bwlday
Splenic hyperplasia was noted for animals not delivered (212, No.: 338 and 341) and subjected to histological examinations because of macroscopic findings.

750 mg/kg bw/day
Foetuses and dilatation of uterus were observed in pregnant animal not delivered (No.: 438).

There were no histological changes in dams with viable foetuses in the high dose group. The ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortex of ovaries in all female animals contained primordial, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.

Non-pregnant and not mated animals

Control

In the only non-pregnant animal, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortex of ovaries contained primordial, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle.

750 mg/kg bw/day
The ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortex of ovaries in all female animals contained primordial, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. In summary: No treatment related alterations were found at the investigation of the male and female reproductive organs, pituitary, and macroscopically altered organs.

750 mg/kg bw/day .

The zonal vacuolisation of hepatocytes in the liver (111 male animal, 100 mg/kg bw/day), the unilateral pyelectasia in the kidney without other pathological lesion (degeneration, inflammation or fibrosis, 212 male control animals, and 1 male animal at 100 mg/kg bw/day ), the foamy cells in the lung (111 male, control) and the hyperplasia in the spleen (300 mg/kg bw/day , two female animals) were considered as individual disorder. These are common alterations in this species and strain. The endometritis in this study was considered as sporadic individual disease.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects related to the substance were noted. The only effects seen were secondary effects related to the high lithium content of the substance.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the 750 mg/kg bw/day group, a slightly higher mortality rate was noted; 25/288
pups were found dead (9) or were cannibalised (15) between postnatal days 0 and 21 in comparison to 10/318 pups in the control group. This was considered not to be related to the toxicity of the substance but due to a secondary effect of the lithium side effects (polydipsia and polyurea) in dams.

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Behaviour (functional findings):

no effects observed

F1 GENERATION
MORTALITY AND CLINICAL SYMPTOMS
In the control, 100 mg/kg bw/day and 300 mg/kg bw/day groups, the mortality of pups was low between postnatal days 0 and 2 1 (1 013 18,6126 1 and 71256, respectively).

In the 750 mg/kg bw/day group, a slightly higher mortality rate was noted. Pups were found dead (9) or were cannibalised (15) between postnatal days 0 and 21.
Percentage of dead male pups was significantly higher than in the control group.
There were no differences between control and test item treated groups in the ratio of genders.
Viability indices and lactation indices were similar in the control, 100 mg/kg bw/day and 300 mg/kg bw/day groups during the 21 postnatal days. In the 750 mg/kg bw/day group, the viability index was slightly less on postnatal day 4.

There were no differences in the litter means of the examined parameters between the
control and dose groups.

In summary: A slightly higher mortality rate of male pups was found at 750 mg/kg bw/day between postnatal day 0 and 4, consequently the viability indices for this period was less. This difference was ascribed to treatment, but was probably related to maternal toxicity (observed as polyuria).

The percentage of non-suckled pups correlated with the percentage of dead or cannibalised (not found) pups at 750 mg/kg bw/day group. Pale, cold pups, congenital absence of tail and haemorrhage on the body were observed sporadically without any dose relation.

BODY WEIGHT
There were no differences in the mean body weight and body weight gain of pups between the experimental groups when calculated either individually or by litter.

DEVELOPMENTAL TESTS
The number and percentage of animals with a negative response in the surface righting reflex and in the suckling ability were comparable in all groups. The number of pups per litter with positive surface righting reflex was slightly less than the control.
The number and percentage of animals with detached pinna (negative response) was higher at 300 mg/kg bw/day and the number and percentage of animals with closed eyes (negative response) was less at 100 mg/kg bw/day . There were no differences in the litter means.

In summary: There was no test item influence on the development of pups. The differences in pinna detachment and eye opening at 300 and 100 mg/kg bw/day , respectively, were considered to be independent from the test item taking into account the normal values of the high dose group.

NECROPSY
In the control group, acute enteritis (115) empty stomach (115) and autolysed organs (115) were observed. The stomach was full of milk (1/5), the lung flotation test was positive in one rat (115) and was negative in another one (115) on postnatal day 0. In the 100 mg/kg bw/day group, autolysed organs (3/5), empty stomach (215) and dark red lungs (115) were found. The lung flotation test was positive in two pups. In the 300 mg/kg bw/day group, autolysed organs (211 l), empty stomach (111 1) were observed. The lung flotation test was negative in seven pups.

In the 750 mg/kg bw/day group, empty stomach (519) and dark red lungs (219) were found. The lung flotation test was positive in nine pups.
In summary: Test item related macroscopic alterations were not found in offspring subjected to gross pathology. Acute enteritis and content of stomach were considered to be individual findings causing death of the pups.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

viability

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects related to the substance were noted. The only effects seen were secondary effects related to the effects caused in dams by the high lithium content of the substance.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Reaktiv Gelb F68072 FW induced polyuria and polydipsia at 750 mg/kg/day dose level on parental generation of CRL:(WI)BR rats during the course of a one generation reproduction toxicity study. This is a reult of the hight lithium content in the test material, as polyuria and polydipsia are common side effects of lithium.
Reproductive performance of males and females were unaffected by the treatment. The mortality of pups was a slightly higher at 750 mg/kg bw/day, as a consequence of maternal lithium effects (polyuria). There was no effect on postnatal development of viable pups.

Executive summary:

The aim of the one-generation reproduction toxicity study was to provide general information concerning the effect of the test item Reaktiv Gelb F68072 FW on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation and lactation (P generation) and on the neonatal morbidity, mortality, growth and development of the offspring (F1 generation) following oral (by gavage) administration. CRL:(WI)BR rats (n=24 males/group and n=25 females/group) were involved in the study in a control and at three dose levels: 100 mg/kg/day, 300 mg/kg/day and 750 mg/kg/day. Treatment was carried out orally once a day in concentrations of 10 mg/ml, 30 mg/ml and 75 mg/ml corresponding to 10 ml/kg body weight volume. All animals of the P generation were treated prior to mating (male animals for 81 days, females for 27 days) and throughout mating. For females, treatment was continued though the gestation and lactation periods up to the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, estrous cycle, mating, and delivery process.

The dams of P generation were allowed to litter, and rear their young up to termination on day 21-23 postpartum. Developmental tests were evaluated on litters (surface righting reflex, suckling, pinna detachment and eye opening). All animals were subjected to gross pathology one day after the last treatment and offspring were sacrificed. Histopathology examination was performed on reproductive organs in the control and high dose groups and on gross lesions in the low and middle dose groups.

There were no test item related effect on the general state and behavior of animals, but an increased urine excretion and water consumption related to the test item was found at 750 mg/kg bw/day group (male and female animals) from day 2 until the termination of the treatment. The body weight, body weight gain of males and females were unaffected at the examined dose levels during the pre-mating period. There was no effect on body weight, body weight gain of dams during gestation and lactation period at the examined dose levels. The mean daily food consumption was higher than the control at 750 mg/kg bw/day dose in male animals and in female animals during the pre-mating and gestation periods. No test item influence on the estrous was found.

There were no test item related alterations in the delivery data of dams when compared with the control value.

Reproductive performance of males and females were unaffected by treatment with Reaktiv Gelb F68072 FW. Gross pathology revealed no alterations due to the effect of test item in P generation. No histological alterations related to the test item effect were found. In the male animals the investigated organs of reproductive system (testes, epididymides, seminal vesicles, prostate, coagulating gland) were histologically normal. In dams, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.

Mortality of pups was slightly higher at 750 mg/kg bw/day group (9% vs. 3% of control value). Body weight, body weight gain and sex ratio of pups were not influenced by treatment with Reaktiv Gelb F68072 FW. There was no effect on postnatal development of pups.

Reaktiv Gelb F68072 FW induced polyuria and polydipsia at 750 mg/kg/day dose level on parental generation of CRL:(WI)BR rats during the course of a one generation reproduction toxicity study. This is a reult of the hight lithium content in the test material, as polyuria and polydipsia are common side effects of lithium.
Reproductive performance of males and females were unaffected by the treatment. The mortality of pups was a slightly higher at 750 mg/kg bw/day, as a consequence of maternal lithium effects (polyuria). There was no effect on postnatal development of viable pups.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

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

A one-generation reproduction toxicity study was performed to provide general information concerning the effect of the test item Reactive Yellow F68072 on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation and lactation (P generation) and on the neonatal morbidity, mortality, growth and development of the offspring (F1 generation) following oral (by gavage) administration. CRL:(WI)BR rats (n=24 males/group and n=25 females/group) were involved in the study in a control and at three dose levels: 100 mg/kg/day, 300 mg/kg/day and 750 mg/kg/day. Treatment was carried out orally once a day in concentrations of 10 mg/ml, 30 mg/ml and 75 mg/ml corresponding to 10 ml/kg body weight volume. All animals of the P generation were treated prior to mating (male animals for 81 days, females for 27 days) and throughout mating. For females, treatment was continued though the gestation and lactation periods up to the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, estrous cycle, mating, and delivery process.
The dams of P generation were allowed to litter, and rear their young up to termination on day 21-23 postpartum. Developmental tests were evaluated on litters (surface righting reflex, suckling, pinna detachment and eye opening). All animals were subjected to gross pathology one day after the last treatment and offspring were sacrificed. Histopathology examination was performed on reproductive organs in the control and high dose groups and on gross lesions in the low and middle dose groups.
There were no test item related effect on the general state and behavior of animals, but an increased urine excretion and water consumption related to the test item was found at 750 mg/kg bw/day group (male and female animals) from day 2 until the termination of the treatment. The body weight, body weight gain of males and females were unaffected at the examined dose levels during the pre-mating period. There was no effect on body weight, body weight gain of dams during gestation and lactation period at the examined dose levels. The mean daily food consumption was higher than the control at 750 mg/kg bw/day dose in male animals and in female animals during the pre-mating and gestation periods. No test item influence on the estrous was found.
There were no test item related alterations in the delivery data of dams when compared with the control value.
Reproductive performance of males and females were unaffected by treatment with Reactive Yellow F68072. Gross pathology revealed no alterations due to the effect of test item in P generation. No histological alterations related to the test item effect were found. In the male animals the investigated organs of reproductive system (testes, epididymides, seminal vesicles, prostate, coagulating gland) were histologically normal. In dams, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.
Mortality of pups was slightly higher at 750 mg/kg bw/day group (9% vs. 3% of control value). Body weight, body weight gain and sex ratio of pups were not influenced by treatment with Reactive Yellow F68072. There was no effect on postnatal development of pups.
Reactive Yellow F68072 induced polyuria and polydipsia at 750 mg/kg/day dose level on parental generation of CRL:(WI)BR rats during the course of a one generation reproduction toxicity study. This is a reult of the hight lithium content in the test material, as polyuria and polydipsia are common side effects of lithium.
Reproductive performance of males and females were unaffected by the treatment. The mortality of pups was a slightly higher at 750 mg/kg bw/day, as a consequence of maternal lithium effects (polyuria). There was no effect on postnatal development of viable pups.

The NOAEL was hence considered to be >= 750 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Reactive Yellow F68072 induced polyuria and polydipsia resulting in slight maternal toxicity at 600 mg/kg bw/day as a consequence of a high content of lithium salt. There were no effects of treatment on the early or late embryonic mortality or in foetal deaths.  No foetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June to 20 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

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
- Species and strain: Crl:(Wi) BR-Wistar Rats
- Source: males: CHARLES RIVER EUROPE LABORATORIES INC., Germany
females: CHARLES RIVERlEUROPElLABORATORIES INC., TOXI-COOP 1103 Budapest, Hungary
- Age at study initiation: Males: from stock of 'experienced' males
Females: 45-55 days
- Weight at study initiation: 179 and 290 g (females)
- Housing: pre-mating and mating period : 1-3 animals/cage
during mating hours : 1 male with 1- 3 females
during pregnancy : 1-4 sperm positive females/cage
Three dams were caged individually
- Diet: Ssniff SM RIM-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance" ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 June To: 24 July 2007

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

The test item was administered at appropriate concentrations prepared with the vehicle. Preparation of test item solution was made using magnetic stirrer. Dose formulations were freshly prepared daily except for five cases when the formulations were prepared 24 or 48 hours before treatment and were kept in refrigerator.
Sampling for analytical control of dosing solutions was made during the first and last week of the treatment period by the Analytical Laboratory of LAB Hungary.

PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 7.5, 20, 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 9151006

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

PRINCIPLE OF THE ANALYTICAL METHOD
Test item content and homogeneity were determined in dosing formulations at two analytical occasions.
6 samples were taken from each test concentration and one from the control.
The samples were properly diluted and analysed by HPLC using UV detection. Concentration of the active ingredient was determined.

EQUIPMENT AND CHEMICALS
APPARATUS
HPLC system: Merck-Hitachi LaChrom HPLC system
D-7000 Interface, No. : 1442- 122
L-7100 HPLC pump, No.: 1516-030
L-7200 Autosampler, No.: 1406-005
L-7400 UV Detector, No.: 1502-017
L-7360 Column oven, No.: 00107295
L-7614 Degasser, No.: 144 12YA0500
Balances: L 2200P Sartorius, Germany, No.:38 100037
BP221 S Sartorius, Germany, No.: 1 18091 17
Ultrasonic bath: Elmasonic S 300 H, Elma No.: 01 08901 05
Water purification system: MILLIPORE, DIRECT 43, FOMNO 73341
Chromatographic conditions:
Detector: UV at 270 nm
Column: LiChrospher 60 RP Select B 5pm 250x4 mm,
No.: 437724
Column temperature: 30 deg C
Mobil Phase: 2.5 g TBAHS was dissolved in 620 ml water and then 280 ml acetonitrile and 100 ml isopropanol were added
Flow: 1.0 ml/min
Injection volume: 20 µl

all concentrations were between 94% and 101% of nominal concentrations

Details on mating procedure:

The oestrus cycle of female animals were examined before pairing. After acclimatisation, the females were paired according to their oestrus cycle to males in the morning for three hours (one male: one to three females) until the number of sperm positive females I group achieved twenty two. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm. The day of mating was regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 2 to 4 animals. Three dams were caged individually.

Duration of treatment / exposure:

day 5 to 19 of gestation

Frequency of treatment:

daily

Duration of test:

START OF THE EXPERIMENT: 1 8 June 2007
END OF IN-LIFE PHASE: 24 July 2007

No. of animals per sex per dose:

22 sperm-positive females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level was selected on the basis of the results of the studies
- Reaktiv Gelb F-68 072 FW Testing for subacute oral toxicity (28 applications within 29 days) in male and female Wistar rats (Hoechst AG report No. 93.0537)
- Draft report 90-Day oral toxicity study of Reaktiv F68072 FW in rats. LAB Report dated 3. Jan. 2007

In the 28 day study with Reaktiv Gelb the daily administration of 600 mg/kg caused adverse clinical symptoms and increased water consumption Furthermore, decreases in serum-chloride and serum-sodium levels were observed. Increases in GPT-activities were evaluated as indicative for an adverse effect on the liver. Additionally, decreases in erythrocyte counts, haemoglobin and haematocrit values as well as increases of liver weight were observed. After administration of 120 mg/kg/day water consumption was still increased in both sexes and GPT acivities were higher compared to controls. The "no observed effect level" was therefore 24 mg/kg body weight.

In the 90 day study conducted in 2006 dose levels of 1000, 250 and 62.5 mg/kg/day were administered.
Polyuria was observed at all dose levels tested. At 1000 mg/kg/day, numbers of erythrocytes, haemoglobin and heamotocrit values and platelet counts were lower than in controls.
In summary a dose level of 250 mg/kg/day was considered to represent a "no toxic effect level".

Therefore, a dose level of 600 mgkg is determined to represent the highest dose level to be tested in the rat embryotoxicity study. A dose level of 600 mg/kg/day is by a factor of
25 higher than the "no toxic effect level" in the 28 day study and by a factor of 2.4 higher than the "no toxic effect level" in the 90 day study.

Maternal examinations:

Clinical Observations
A careful clinical observation was made after dosing at least once a day and a cage side clinical observation was made in the afternoon. Individual observation included the check of behavior and general condition.
Checking for death was made at least once daily.

Body Weight
The body weight of the male animals was not measured.
The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).
Corrected body weight was calculated on the day 20 of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

Food Consumption
The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

Water consumption
The water consumption was measured each day from gestation day 0 to 20 with reweighing the non-consumed water with a glass-measure (accuracy: 1 ml).

Examination for Sign of Implantation
On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses.

NECROPSY
All sperm positive females were sacrificed by CO2 gas anesthesia followed by cervical dislocation on day 20 of gestation. The abdomen was opened, the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus and its content gross pathology of dams' viscera was performed.
Organs and tissues with undiagnosed macroscopic findings were examined histologically. All these organs were fixed in 10 % buffered formalin solution after necropsy. For histological examinations organs and tissues were embedded into paraffin after dehydration.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live fetuses, early and late embryonic death and fetal death were counted. Animals with unambiguous implantation sites, but not fetuses have been found, were considered as pregnant.

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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Fetuses were removed from the opened uterus, viability assessments were performed and after the umbilicus was cut euthanasia of the viable fetuses was made by CO2 gas.
Each live fetus was weighed individually (accuracy 0.01 g), and was subjected to external examination. The placentas were examined externally. The gender of fetuses was determined according to the anogenital distance.
Thereafter the fetuses were individually identified and about the half of each litter was subjected to visceral examination and the other half for skeletal examination. In case of fetuses subjected to visceral examination the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. In case of fetuses subjected to skeletal examination the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid – ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett's test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. A foetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of all control foetuses.

Dams were excluded from statistical analysis as follows:
- non pregnant females:
Control: No.: 139, 146,214
75 mglkg: No.: 114,209
200 mg/kg: No.: 181,208
600 mg/kg: No.: 122

- female with no implantation, with corpora lutea: Control: 168
- females with 5 or less implantation: 75 mg : No.: 207; 600 mg : No.: 182
- females with placental inflammation or diffuse necrosis: 75 mg : No.: 184,103; 600 mg : No.: 204
- female excluded due to technical error (error in determining the day 0 of pregnancy): 200 mg : 137

The following data were evaluated by an appropriate statistical method (Bartlett, ANOVA - Duncan, Kruskal-Wallis - Mann-Whitney U test, chi2):

PREGNANT FEMALES
- Mortality
- (Corrected) body weight, body weight gains, gravid uterine weight, food consumption, water consumption: mean +/- S.D.
- Gross pathology observation

NECROPSY AND CESAREAN SECTION DATA
- Number of corpora lutea, number of implantations, number and percent of live fetuses, fetal weight, number and percent of intrauterine mortality: mean +/- S.D.

Indices:

- Pre-implantation loss: Number of corpora lutea - Number of implantations x 100 / Number of corpora lutea
- Post-implantation loss: Number of implantations - Number of live fetuses - x 100 /Number of implantations

FETUSES
- Sex distribution: Number of Male (Female) fetuses x 100 / Number of fetuses
- External abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses
- Visceral abnormalities/litter: Number of fetuses with abnormality - x 100 / Number of fetuses
- Skeletal abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses

Historical control data:

Yes, within LAB Research Inc, Hungary's historical range.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Observation of polyuria (wet bedding) was recorded for the dams in all cages in the 600 mg/kg dose group during the treatment period. It was considered to be a secondary maternal toxicity resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The water consumption of dams was significantly higher than controls in the 600 mg/kg dose group during the treatment period (between gestation days 5- 1 1, 1 1 - 17 and 1 7-20). This was considered to be an effect resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Early death and consequential postimplation loss was statistically significant higher in the 75 mg/kg and 200 mg/kg dose groups compared to the control group. A statistically significant higher total intrauterine mortality in the 200 mg/kg dose group was also a consequence of the high postimplation loss level, however, early embryonic death, postimplantation loss and total intrauterine mortality were below the control level in the 600 mg/kg dose group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

CLINICAL SYMPTOMS, MORTALITY
There were no dead animal in any group. Observation of polyuria (wet bedding) was recorded for the dams in all cages in the 600 mg/kg dose group during the treatment period. It was considered to be a sign of treatment related maternal toxicity. Piloerection was recorded for one single dam (No.: 184) in the 75 mg/kg dose group, this was not attributed to the administration of the test item.

BODY WEIGHT, BODY WEIGHT GAIN, GRAVID UTERINE WEIGHT, AND CORRECTED BODY WEIGHT
The body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain of dams were at the same level in the control and in the Reaktiv Gelb F68072 FW treated groups.

FOOD CONSUMPTION
There was no difference in the food consumption of dams in the experimental groups.

WATER CONSUMPTION
The water consumption of dams was significantly higher than controls in the 600 mg/kg dose group during the treatment period (between gestation days 5- 1 1, 1 1 - 17 and 1 7-20). The 75 mg/kg and 200 mg/kg dose groups were unaffected.

NECROPSY
At necropsy there were no treatment related gross pathology alterations seen. Reddish-mottled lungs observed in one dam (No.: 198) in the control group, one dam (No.: 120) in the 75 mg/kg dose group, point-like haemorrhages on the lungs for one dam (No.: 143) in the 600 mg/kg dose group were related to euthanasia and exsanguination. Pale liver and dark coloured fluid in the uterus were recorded for the same dam (No.: 184) in the 75 mg/kg dose group and was not attributed to the treatment. A slightly enlarged spleen was recorded for one dam (No.: 204) in the 600
mg/kg dose group, this was not considered to be treatment related. Orange coloured content in the stomach recorded for 9 females (No.: 160, 1 12,2 19, 107, 1 15, 143, 149, 166, 190, 122) in the 600 mg/kg dose group was related to the orange colour of the test item Reaktiv Gelb F68072 FW.

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

All effects seen were considered to be secondary effects due to the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two malformations out of 232 foetuses were found in the control group, one hydrops fetalis and one foetus with absent tail and with filum terminale hanging out.
One malformed foetus out of 235 was found in the 600 mg/kg dose group also with
the malformation mentioned above. These malformations are regarded as spontaneous and unrelated to treatment. There were no other malformations found during the external examination.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

REPRODUCTION DATA, INTRAUTERINE MORTALITY, VIABLE FOETUSES AND THEIR SEX DISTRIBUTION

The intrauterine mortality or sex distribution of foetuses was most probably not influenced by the treatment. There was no difference between the experimental groups in the number of corpora lutea or implantations.

Early death and consequential postimplation loss was statistically significant higher in the 75 mg/kg and 200 mg/kg dose groups compared to the control group. A statistically significant higher total intrauterine mortality in the 200 mg/kg dose group was also a consequence of the high postimplation loss level, however, early embryonic death, postimplantation loss and total intrauterine mortality were below the control
level in the 600 mg/kg dose group. There were statistically significant fewer male foetuses and more female foetuses in the 75 mg/kg dose group compared with the other experimental groups, however, the sex distribution was within the historical background range in this dose group.

In summary, there were statistical differences in some parameters, but there was no dose-response and all values were considered to be normal. There were no effects of treatment on these parameters.

BODY WEIGHT OF FOETUSES
The average body weight of foetuses was similar in all groups.

FOETAL EXAMINATION
External Examination:
Two malformations out of 232 foetuses were found in the control group, one hydropsfetalis and one foetus with absent tail and with filum terminale hanging out. One malformed foetus out of 235 was found in the 600 mg/kg dose group also with the malformation mentioned above. These malformations are regarded as spontaneous and unrelated to treatment. There were no other malformations found during the external examination.
The incidence of body weight retarded foetuses was significantly higher in the 200 mg/kg group but the majority were all in one litter. There was a consequent higher incidence of foetuses with external abnormalities in this group. The incidence of external abnormalities in the 600 mg/kg dose group was lower than the control level. The type and incidence of external foetal observations was within the historical background range, there were no treatment related effects seen at external examination.

The incidence of placentas with fibrinoid degeneration (an inflammatory reaction) was significantly higher in the 600 mg/kg dose group compared with the other experimental groups. In 11 of 15 cases the change was seen in two litters the other 4 cases appeared sporadically. Small placentas were found in 6 out of 235 foetuses in the 600 mg/kg dose group. The differences in incidence of placental observations is not considered to be a foetal effect of treatment, it is equivocal as to being an effect of treatment, it may reflect maternal toxicity.
There was a sporadic incidence of fused, pale, oedemateous, dark brown discoloured placentas found in the experimental groups, without relationship to treatment.

Visceral Examination:
At visceral examination the incidence of abnormalities was higher in the control group than in the Reaktiv Gelb F68072 FW treated groups.
Four foetuses with malformations out of 1 15 were found in the control group. Three of these foetuses were multiple malformed and one foetus had situs inversus cordis. One of the three multiple malformed foetuses had one lung lobe in each side, stomach and intestines on the right side (situs inversus abdominalis).

The second of the three multiple malformed foetuses had one lung lobe on each side, aorta turned to the right, truncus pulmonalis turned to the left, right subclavian artery, right carotid artery and left carotid artery originated from the top of aortic arch, left subclavian artery originated from truncus pulmonalis, aorta turned behind oesophagus to the left, septum membraneceum was absent, (VSD), aorta was overriding the septal
defect, stomach and intestines were on the right side (situs inversus abdominalis), duodenum was narrowed by vena portae.The third multiple malformed foetus (one of the two foetuses with caudal regression syndrome) had small and empty stomach, absent gonads, kidneys close to each other and fused adrenals.
There was one malformed foetus with a malpositioned kidney out of 101 in the 75 mg/kg dose group. One malformed foetus with yellowish discolouration in the lens (histopathologically central cataracta) out of 108 foetuses was found in the 200 mg/kg dose group. Two malformations were recorded out of 120 foetuses in the 600 mg/kg dose group. One foetus had a malpositioned right kidney and the other foetus was multiple malformed with absent gonades and fused kidneys. This multiple malformed foetus also had caudal regression syndrome indicated by the observations of external and skeletal examinations (there was a similar foetus in the control group).
Variations unrelated to treatment observed included short and extremely short brachocephalic trunk, left common carotid artery originated close to brachiocephalic trunk, dilated renal pelvis and dilated third and lateral brain. The incidence of dilated renal pelvis was the highest in the 600 mg/kg dose group, but it was within the historical control range.
All types of visceral abnormalities and variations described are present in the historical database of our Laboratory or are described in teratology literature as spontaneous changes. There were no effects of treatment on the incidence of any visceral variations.

Skeletal Examination:
There were more skeletal malformations found in the control group than in the test item treated groups. There were no types of malformation present with a significantly higher incidence in the 600 mg/kg dose group than in the control group. There was one foetus out 1 19 with malformed vertebrae in the control group (the one of the two foetuses found during the study with caudal regression syndrome). This foetus had four cervical vertebrae, the other vertebrae and the ribs were absent. There were no foetus with malformed vertebrae in the 75 mg/kg or the 200 mg/kg dose
groups. Malformed vertebrae were recorded for one out of 1 16 foetuses in the 600 mg/kg dose group also with caudal regression syndrome. This foetus had similar skeletal abnormalities as the above mentioned one, 4 cervical vertebrae were present, fused bones instead of thoracic III and lumbar VI vertebrae and only the first and second ribs were present.

Short and/or bent clavicula and/or scapula were recorded for 4 foetuses in the control group out of 119, for 3 out of 98 in the 75 mg/kg dose group, for 2 out of 109 in the 200 mg/kg dose group and for 3 out of 1 16 in the 600 mg/kg dose group. Short and/or thickened humerus was observed in 2 foetuses in the control group, none in the 75 mglkg dose group, in 3 in the 200 mg/kg dose group and in 2 in the 600 mg/kg dose group.

Short and/or bent radius and/or ulna were found only in two foetuses in the control group.
Short and/or bent osilei was found only in one foetus in the 200 mg/kg dose group.
Short and/or bent femur and fibula were recorded for one control foetus.

At skeletal examination there were a range of variations in examined foetuses, but these variations showed no dose response and were unrelated to treatment. These variations included incomplete ossification of the skull, 2 or less than 2 sternal bodies, 13'h rib short, markedly wavy ribs with or without incomplete ossification, bipartite thoracic vertebrae, irregular calcification or not ossified thoracic, lumbar or sacral vertebral arches, 14 thoracic, 5 lumbar vertebrae, not ossified or incompletely ossified sacral vertebra 11, 2 or less or asymmetric metacarpus, 3 or less or asymmetric metatarsus, incomplete ossification of clavicula and slightly short clavicula and scapula, irregular periosteal ossification on humerus and/or ulna or femur. Incomplete ossification of clavicula, slightly short or bent clavicula or scapula. Incomplete or unossified os pubis.
Incomplete ossification or non-ossified os ischii was not present in the control group but it was recorded in the Reaktiv Gelb F68072 FW treated groups. However, in the most recent comparable study in this laboratory, the control incidence was 6/121 foetuses (and for reduced ossification of the os pubis it was 18/121 foetuses). Hence, taking into account the historic control data for incomplete ossification or non-ossified
os ischii, the statistical difference is not considered to be treatment related.

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

All effects seen were considered to be secondary effects due to maternal toxicity resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Slight maternal toxicity was observed at 600 mg/kg bw/day only resulting from a high content of lithium salt. There were no effects of treatment on the early or late embryonic mortality or in fetal deaths. No fetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations.

Executive summary:

Groups of 22 sperm-positive female Crl:(Wi)BR-Wistar rats were treated with REAKTIV GELB F68072 FW orally in one control group and three dose levels of 75, 200, and 600 mg/kg/day with 10 ml/kg treatment volume from day 5 up to and including day 19 post coitum. Controls were given vehicle alone, the vehicle was distilled water.

Rats were examined daily for viability and clinical signs. Body weight was recorded between Days 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. Food consumption was determined on Days 0-3, 3-5, 5-8, 8-1 1, 11-14, 14-17 and 17-20 of gestation, water consumption was measured each day from gestation day 0 to 20. Caesarean section and gross-pathology were performed on day 20 of pregnancy. Implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea in each ovary were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally. About half of each litter was subjected to visceral examination and the other half to skeletal examination (with double staining). All abnormalities found during the fetal examinations were recorded.

There was no maternal mortality in any group. Observation of high water intake and polyuria (wet bedding) was recorded for the dams in each cage in the 600 mg/kg dose group during the treatment-period as a treatment related maternal clinical sign. There were equivocal statistical differences in the incidence of placentas with fibrinoid degeneration at 600 mg/kg/day, which may be related to maternal toxicity but was without any fetal developmental effects. There were no treatment related clinical signs in the other experimental groups.

The test item had no effect on the body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain or food consumption of dams.

At necropsy no treatment related gross pathology alterations were seen. The intrauterine mortality or sex distribution of fetuses was not influenced by the treatment. There was no difference between groups in the number of corpora lutea or implantations.

The body weight of fetuses was similar in all experimental groups. There were no abnormalities attributable to the treatment at external, visceral or skeletal examinations.

In Conclusion:

Slight maternal toxicity was observed at 600 mg/kg/day only. There were no effects of treatment on the early or late embryonic mortality or in fetal deaths.

No fetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations.

Under the conditions applied in this study the observations made in the dams treated with Reaktiv Gelb F68072 FW, and their fetuses the following no observable-adverse-effect levels were derived:

NOAEL maternal toxicity: 200 mg/kg bw/day

NOAEL developmental toxicity: 600 mg/kg bw/day

NOAEL teratogenicity: 600 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

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

Groups of 22 sperm-positive female Crl:(Wi)BR-Wistar rats were treated with Reactive Yellow F68072 orally in one control group and three dose levels of 75, 200, and 600 mg/kg/day with 10 ml/kg treatment volume from day 5 up to and including day 19 post coitum. Controls were given vehicle alone; the vehicle was distilled water. Rats were examined daily for viability and clinical signs. Body weight was recorded between Days 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. Food consumption was determined on Days 0-3, 3-5, 5-8, 8-1 1, 11-14, 14-17 and 17-20 of gestation, water consumption was measured each day from gestation day 0 to 20. Caesarean section and gross-pathology were performed on day 20 of pregnancy. Implantations, early and late resorptions, live and dead foetuses in each uterine horn and the number of corpora lutea in each ovary were recorded. Each foetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally. About half of each litter was subjected to visceral examination and the other half to skeletal examination (with double staining). All abnormalities found during the foetal examinations were recorded.

There was no maternal mortality in any group. Observation of high water intake and polyuria (wet bedding) was recorded for the dams in each cage in the 600 mg/kg dose group during the treatment-period as a treatment related maternal clinical sign. This is a secondary effect due to the high lithium content, as polydipsia and polyurea are common side effects of treatment with lithium. There were equivocal statistical differences in the incidence of placentas with fibrinoid degeneration at 600 mg/kg/day, which may be related to maternal toxicity but was without any foetal developmental effects. There were no treatment related clinical signs in the other experimental groups. The test item had no effect on the body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain or food consumption of dams. At necropsy no treatment related gross pathology alterations were seen. The intrauterine mortality or sex distribution of foetuses was not influenced by the treatment. There was no difference between groups in the number of corpora lutea or implantations. The body weight of foetuses was similar in all experimental groups. There were no abnormalities attributable to the treatment at external, visceral or skeletal examinations.

In conclusion, slight maternal toxicity was observed at 600 mg/kg bw/day only resulting from a high content of lithium salt. There were no effects of treatment on the early or late embryonic mortality or in foetal deaths.  No foetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations. The NOAEL was therefore considered  to be >= 600 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

All effects seen were secondary effects of polydipsia and polyuria caused by a high lithium salt content in the testing material. Polydipsia and polyuria are common side effect of treatment with lithium. This effect is not relevant for humans, as the high lithium intake will not occur during use of the substance of the dyed material.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for effects is therefore required.