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Toxicity to reproduction

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March - 31 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
Reproduction/Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11. March - 31 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Rheinland-Pfalz, 21.03.2017
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0018279326
- Content: 100.0 g/100 g - 0.7 g/100 g (water) = 99.3 g/100 g
- Expiry date: Dec 2019
- Purity test date: Dec 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Analytical verifications of the stability of the test substance in the diet for a period of 32 days at room temperature were verified before the study was initiated with a comparable batch
- The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required quantity of test substance was weighed in a beaker depending on the dose group
and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food,
depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): with diet
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Animals were free from any clinical signs of disease
- Age at study initiation: 11-12 weeks (males); 10 weeks (females)
- Housing: individually during study period (during overnight matings male and female mating partners were housed together; pregnant animals and their litters were housed together until PND 13); grouped (up to 5 animals per sex and cage) during pretreatment
- Diet: ad libitum (from the day of supply to the day before necropsy); ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: about 3 weeks

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
Analytical verification of doses or concentrations:
yes
Remarks:
LC/ESI-MS
Details on analytical verification of doses or concentrations:
At the beginning (during premating), once during gestation and once during lactation of the
study each 3 samples were taken from the lowest and highest concentration for potential
homogeneity analyses. These samples were used as a concentration control at the same time.
At the time points mentioned above additionally one sample from the mid concentration was
taken for concentration control analysis.

Averaged recovery rates ranged from 80 to 119 %.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- A vaginal smear was prepared after each mating and examined for the presence of sperm.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
Duration of treatment / exposure:
males: 35 days; females: 59 days
The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:
continuously (via diet)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
for females during lactation: 500 ppm;
= mean intake of approx. 54 mg/kg bw/d (males), approx. 77 mg/kg bw/d (females)
Dose / conc.:
4 000 ppm (nominal)
Remarks:
for females during lactation: 2000 ppm;
= mean intake of approx. 208 mg/kg bw/d (males), approx. 292 mg/kg bw/d (females)
Dose / conc.:
12 000 ppm (nominal)
Remarks:
for females during lactation: 6000 ppm;
= mean intake of approx. 619 mg/kg bw/d (males), approx. 901 mg/kg bw/d (females)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: During the lactation period the test substance concentrations in the diet of the
F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food
consumption data maintained the dams at the desired target doses of the test item during this period of increased food intake.

For details regarding the dosing see table 2 in "Any other information on materials and methods"
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (for mortality, any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice with the following exceptions for the females:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 10, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1), 4, 7, 10 and 13.
The body weight change of the animals was calculated from these results.
Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-10, 10-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: No
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
Pub examination: External examination and visceral examination, pub organs were assessed macroscopically;
Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone
measurement. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution
Statistics:
see table 1 in "Any other information on materials and methods"
Indices:
Postimplantation loss, live birth index (for detailed formulas see "Any other information on materials and methods")
Historical control data:
Historical control data to allow comparison with concurrent controls were provided for the following parameters: maternal body weights, mating data, delivery data, litter data, sex ratio, pup weights, anogenital distance and age index, pup necropsy observations
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights/body weight gain of low- and mid-dose F0 parental females were comparable to the concurrent control values during the entire study period.
The mean body weights of the high-dose females (12000 ppm) were below the concurrent control throughout the study, the difference was statistically significant on GD 20 and PND 1 - 4 (about 6% and 10%, respectively).
The body weight change of the high-dose females was statistically significantly below the concurrent control values during GD 0 - 20 (about 18%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose (12000 ppm) F0 females was consistently below the concurrent control, the difference was statistically significant during during GD 14 - 20 (about 9%).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not specified
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:
The mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal
biological variation into account (12.8, 11.8, 11.8 and 12.2 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the post-implantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.3, 11.4, 10.9 and 11.7 pups/dam in test groups 0 - 3, respectively).
live-birth index: With the exception of 2/10, 1/10, 1/1o female rats the index was 100%
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
12 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: No maternal developmental toxicity observed up to and including the highest dose tested.
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
12000 ppm: Decreased pup body weights during PND 7 - 13 (12 - 13% below control), decreased pup body weight gain during PND 7 - 13 (13 - 15% below control), overall the high-dose pups
gained 14% less weight during lactation days 1 - 13 (see table 3).
The mean pup body weights and pup body weight change of the low- and mid-dose male and female pups were comparable to the concurrent control values throughout lactation days 1 -
13. One female runt was seen in the control, one male runt was seen in test group 2, and four male as well as seven female runts were seen in test group 3.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Pups at PND13 (test groups 11, 12 and 13; 1000, 4000 and 12000 ppm) showed no treatment-related alterations of T4 and TSH levels.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 3: Pub body weights

 

0 ppm

1000 ppm / 500 ppm

4000 ppm / 2000 ppm

12000 ppm / 6000 ppm

Day 1

6.7

6.9

6.8

6.3

Day 4

10.6

11.0

10.9

9.6

Day 7

17.5

17.6

17.7

15.6 *

Day 13

33.4

33.1

33.2

29.2 **

Statistic Profile = Dunnett test (two-sided), * p<= 0.05, ** p <= 0.01

Conclusions:
In this Reproduction/Developmental Toxicity Screening Test exposure to the test substance resulted in signs of systemic toxicity at a concentration of 12000 ppm, such as reduced food consumption and impaired body weight development. Thus, the no observed adverse effect level (NOAEL) for maternal toxicity was 4000 ppm. The no observed adverse effect level (NOAEL) for developmental toxicity was 4000 ppm, based on lower pup weights at 12000 ppm.
Executive summary:

In this Reproduction/Developmental Toxicity Screening Test, performed in accordance to OECD TG 421 and in compliance with GLP, the test substance was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations (0 ppm, 1000 ppm, 4000 ppm, and 12000 ppm).

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions. The overall mean dose of test substance throughout all study phases was approx. 54 mg/kg body weight/day (mg/kg bw/d) in the 1000 ppm group, approx. 208 mg/kg bw/d in the 4000 ppm group and approx. 619 mg/kg bw/d in the 12000 ppm group (males) and approx. 77 mg/kg bw/d in the 1000 ppm group, approx. 292 mg/kg bw/d in the 4000 ppm group and approx. 901 mg/kg bw/d in the 12000 ppm group (females).

The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Signs of general systemic toxicity were observed in maternal animals exposed to 12000 ppm taking reduced food consumption and impaired body weight development into account. Reduction of food consumption and body weight gain was particularly distinct in parental females towards the end of gestation and during early lactation. Clinical pathology revealed no treatment-related adverse effects on thyroid hormones in any of the dose groups. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Pup weights (average 12-13% below control) and weight gain (average 14% below control) were significantly lower in in the high-dose group (12000 ppm). In addition, a higher number of runts was noted in this test group. Most part of this developmental delay can certainly be attributed to the distinct maternal toxicity which was evident by reduced food consumption/body weight gain of the dams, in particular towards the end of gestation and during lactation. However, independently of the primary or secondary nature of the reduced pup weights the low magnitude of the growth disturbance suggests that it is likely for the pups to make a recovery over time. Thus, this effect was considered to be treatment-related and adverse, but of low toxicological significance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Rheinland-Pfalz, 21.03.2017
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate
EC Number:
942-523-5
Cas Number:
2156594-77-1
Molecular formula:
Unspecified
IUPAC Name:
tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0018279326
- Content: 100.0 g/100 g - 0.7 g/100 g (water) = 99.3 g/100 g
- Expiry date: Dec 2019
- Purity test date: Dec 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Analytical verifications of the stability of the test substance in the diet for a period of 32 days at room temperature were verified before the study was initiated with a comparable batch
- The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required quantity of test substance was weighed in a beaker depending on the dose group
and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food,
depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): with diet

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected
since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Animals were free from any clinical signs of disease
- Age at study initiation: 11-12 weeks (males); 10 weeks (females)
- Housing: individually during study period (during overnight matings male and female mating partners were housed together; pregnant animals and their litters were housed together until PND 13); grouped (up to 5 animals per sex and cage) during pretreatment
- Diet: ad libitum (from the day of supply to the day before necropsy); ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: about 3 weeks

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 1 minute. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- A vaginal smear was prepared after each mating and examined for the presence of sperm.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
Analytical verification of doses or concentrations:
yes
Remarks:
LC/ESI-MS
Details on analytical verification of doses or concentrations:
At the beginning (during premating), once during gestation and once during lactation of the
study each 3 samples were taken from the lowest and highest concentration for potential
homogeneity analyses. These samples were used as a concentration control at the same time.
At the time points mentioned above additionally one sample from the mid concentration was
taken for concentration control analysis.

Averaged recovery rates ranged from 80 to 119 %.
Duration of treatment / exposure:
males: 35 days; females: 59 days
The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:
continuously (via diet)
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm (nominal)
Remarks:
for females during lactation: 500 ppm;
= mean intake of approx. 54 mg/kg bw/d (males), approx. 77 mg/kg bw/d (females)
Dose / conc.:
4 000 ppm (nominal)
Remarks:
for females during lactation: 2000 ppm;
= mean intake of approx. 208 mg/kg bw/d (males), approx. 292 mg/kg bw/d (females)
Dose / conc.:
12 000 ppm (nominal)
Remarks:
for females during lactation: 6000 ppm;
= mean intake of approx. 619 mg/kg bw/d (males), approx. 901 mg/kg bw/d (females)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: During the lactation period the test substance concentrations in the diet of the
F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food
consumption data maintained the dams at the desired target doses of the test item during this period of increased food intake.

For details regarding the dosing see table 2 in "Any other information on materials and methods"
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (for mortality, any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice with the following exceptions for the females:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 10, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1), 4, 7, 10 and 13.
The body weight change of the animals was calculated from these results.
Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-10, 10-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: No

OTHER:

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving adult males at termination
- Parameters checked: Total thyroxine (T4), Thyroid stimulating hormone (TSH)
Oestrous cyclicity (parental animals):
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in parental males:
testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides and testes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (Standardization of litters was not performed in litters with ≤ 8 pups)
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number (on PND 0) and sex of pups, stillbirths, live births, macroscopically evident changes, clinical symptoms (including gross-morphological findings; at least once daily), postnatal mortality (at least once daily), body weight (on PND 1 and PND 4 (before standardization), anogenital distance (AGD) (on PND 1), presence of nipples/areolae in male pups (on PND 13), blood thyroid hormone concentrations (on PND 4 (surplus pups after standardization) and PND 13 (one selected male and one female pup per litter), gross necropsy (at sacrifice (PND 4 (surplus pups after standardiation), PND 13))

GROSS EXAMINATION OF DEAD PUPS
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on study day 35 after start of test substance administration
- Maternal animals: All surviving animals, on study day 59 after start of test substance administration

GROSS NECROPSY
- Special attention was given to the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix

HISTOPATHOLOGY
- Fixation of the following organs was followed by histotechnical processing, examination by light microscopy:
1. Testis
2. Epidymides
3. Ovaries

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required)) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
- All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

GROSS NECROPSY
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.

HISTOPATHOLOGY
- Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.
Statistics:
see table 1 in "Any other information on materials and methods"
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm were detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating index, fertility index (for formulas see "Any other information on materials and methods")
Offspring viability indices:
- viability index, survival index (for formulas see "Any other information on materials and methods")

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights/body weight gain of low- and mid-dose F0 parental males and females were comparable to the concurrent control values during the entire study period.
The mean body weights of the high-dose F0 parental males (12000 ppm) were below the concurrent control throughout the study although the difference did not gain statistical
significance. The mean body weights of the high-dose females (12000 ppm) were below the concurrent control throughout the study, the difference was statistically significant on GD 20 and PND 1 - 4 (about 6% and 10%, respectively).
The body weight change of the high-dose males was statistically significantly below the concurrent control values during in-life days 0 - 7 and 0 - 28 (about 98% and 45%, respectively).
The body weight change of the high-dose females was statistically significantly below the concurrent control values during GD 0 - 20 (about 18%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose (12000 ppm) F0 males was consistently below the concurrent control, the difference was statistically significant during in-life days 0 - 7 (about
14%). Food consumption of the high-dose (12000 ppm) F0 females was consistently below the concurrent control, the difference was statistically significant during during GD 14 - 20 (about 9%).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
In parental males (test groups 1, 2 and 3; 1000, 4000 and 12000 ppm) no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 / 3.9 / 3.9 and 3.9 days in test groups 0 - 3, respectively.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The female/male mating index was 100% in all test groups
the female/male fertility index was 100% all test groups
The gestation index was 100% in all test groups

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
fertility and reproduction
Effect level:
12 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed up to and including the highest dose tested.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weights of the high-dose pups were statistically significantly below the concurrent control values during PND 7 - 13 (12-13%). Accordingly, mean pup body weight
change of the high-dose male and female pups were statistically significantly below the concurrent control values during PND 7 – 13. Overall, the high-dose pups gained 14% less weight during lactation days 1 - 13.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as dilated ureter and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all
these findings were not considered to be associated to the test substance
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
In male and female pups at PND 13 no treatment-related alterations of T4 and TSH levels were observed.
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 100% / 99.2% / 93% and 97.1% in test groups 0 - 3.
The survival index indicating pup survival during further course of lactation (PND 4 - 13) was 100% in all test groups

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 3: Mean body weights for male and female rats

Mean body weight [g]: Sex: Male- Phase: In-life

 

0 ppm

1000 ppm

4000 ppm

12000 ppm

Day 0

371.1

370.2

368.7

368.5

Day 7

383.9

382.2

378.6

368.7

Day 13

388.8

387.8

380.7

375.4

Day 21

396.7

395.2

388.3

380.9

Day 28

404.3

402.7

395.5

386.9

 

Mean body weight [g]: Sex: Female- Phase: In-life

 

0 ppm

1000 ppm

4000 ppm

12000 ppm

Day 0

224.6

222.8

221.8

223.4

Day 7

226.8

227.4

227.4

225.0

Day 13

234.1

234.1

228.8

228.0

 

Mean body weight [g]: Sex: Female- Phase: Gestation

 

0 ppm

1000 ppm

4000 ppm

12000 ppm

Day 0

229.3

227.4

227.3

228.5

Day 7

256.4

253.8

254.7

253.0

Day 10

266.2

263.0

262.4

259.5

Day 14

284.0

279.0

278.2

273.8

Day 20

346.6

337.0

334.8

324.9 *

 

Mean body weight [g]: Sex: Female- Phase: Lactation

 

0 ppm

1000 ppm / 500 ppm

4000 ppm / 2000 ppm

12000 ppm / 6000 ppm

Day 1

263.8

256.4

258.4

238.3 **

Day 4

276.3

272.6

272.6

259.9 *

Day 7

277.8

278.6

281.4

269.7

Day 10

296.5

293.8

293.5

282.5

Day 13

300.0

296.2

297.7

286.0

 

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01

Table 4: Summary Live pubs/litter

 

0 ppm

1000 ppm / 500 ppm

4000 ppm / 2000 ppm

12000 ppm / 6000 ppm

Pups delivered

123

114

109

117

Found dead [pub]/Dead

0

0

0

2

Stillborn /Dead

1

4

1

1

cannibalized [pup] / Dead

0

1

6

0

Pubs surviving day 0 -4

122

109

102

114

From PND 5 - 13 no pub mortality was observed

Applicant's summary and conclusion

Conclusions:
In this Reproduction/Developmental Toxicity Screening Test exposure to the test substance resulted in signs of systemic toxicity at a concentration of 12000 ppm, such as reduced food consumption and impaired body weight development. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 4000 ppm for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 12000 ppm, the highest tested dose. The no observed adverse effect level (NOAEL) for developmental toxicity was 4000 ppm, based on lower pup weights at 12000 ppm.
Executive summary:

In this Reproduction/Developmental Toxicity Screening Test, performed in accordance to OECD TG 421 and in compliance with GLP, the test substance was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations (0 ppm, 1000 ppm, 4000 ppm, and 12000 ppm).

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 35 days under ambient conditions. The overall mean dose of test substance throughout all study phases was approx. 54 mg/kg body weight/day (mg/kg bw/d) in the 1000 ppm group, approx. 208 mg/kg bw/d in the 4000 ppm group and approx. 619 mg/kg bw/d in the 12000 ppm group (males) and approx. 77 mg/kg bw/d in the 1000 ppm group, approx. 292 mg/kg bw/d in the 4000 ppm group and approx. 901 mg/kg bw/d in the 12000 ppm group (females).

The duration of treatment covered a 5 weeks in-life period (males) including 4 days mating (mating pairs were from the same test group) as well as a 2-weeks in-life period (females), 4 days mating period, the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

Signs of general systemic toxicity were observed in parental animals exposed to 12000 ppm taking reduced food consumption and impaired body weight development into account. Reduction of food consumption and body weight gain was particularly distinct in parental females towards the end of gestation and during early lactation. Clinical pathology revealed no treatment-related adverse effects on thyroid hormones in any of the dose groups. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility, reproductive performance and delivery were not impaired by test-substance up to a concentration of 12000 ppm, as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups. Viability and survival indices as indicators for pup mortality in lactation were also not significantly altered.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 4000 ppm for male and female Wistar rats. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 12000 ppm, the highest tested dose.

Pup weights (average 12-13% below control) and weight gain (average 14% below control) were significantly lower in in the high-dose group (12000 ppm). In addition, a higher number of runts was noted in this test group. Most part of this developmental delay can certainly be attributed to the distinct maternal toxicity which was evident by reduced food consumption/body weight gain of the dams, in particular towards the end of gestation and during lactation. However, independently of the primary or secondary nature of the reduced pup weights the low magnitude of the growth disturbance suggests that it is likely for the pups to make a recovery over time. Thus, this effect was considered to be treatment-related and adverse, but of low toxicological significance.