Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
determination of additional parameters as required by OECD 416
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Potassium naphthalene-1-sulphonate
EC Number:
253-850-1
EC Name:
Potassium naphthalene-1-sulphonate
Cas Number:
38251-26-2
IUPAC Name:
potassium naphthalene-1-sulfonate
Constituent 2
Reference substance name:
Naphthalenesulfonic acids, potassium salts
IUPAC Name:
Naphthalenesulfonic acids, potassium salts
Details on test material:
- Name of test material (as cited in study report): Naphthalenesulfonic acids, potassium salts
- Physical state: solid
- Analytical purity: 99.9 % (batch 1), 98.6 % (batch 2) and 98.2 % (batch 3)
- Lot/batch No.: 02/0041-1, 02/0041-2, 02/0041-3
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: (P) 5 wks; (F1) not specified
- Weight at study initiation: (P) Males: 101.5-130.1 g; Females: 89.4-109.7 g; (F1) Males: not specified; Females: not specified
- Housing: housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- 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 / DIET PREPARATION:
For each concentration (with exception of the control food), the test substance was
weighed out and thoroughly mixed with a small amount of diet in a beaker. Subsequently,
a premix was prepared in a food processor emulsifier (Dito Sama (K55 E) Kutter ;
Aubusson, France) by adding an appropriate amount of dry diet and mixing for about 3
minutes . Then appropriate amounts of dry diet, depending on the dose group, were added
to this premix in order to obtain the desired concentrations, and mixing was carried out for
about 10 minutes in a laboratory mixer (Ruberg Mischtechnik KG, Paderborn, Germany).
Dietary concentrations were adjusted throughout the study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: over night for a maximum of 2 weeks
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the diet over 53 days at room temperature was demonstrated prior to the study. This exceeded the interval from
diet preparation to the end of the feeding period.
Homogeneity and concentration control analyses were carried out at the beginning and towards the end of the premating period, as well as during the period after weaning. At least one analysis of each of the test substance preparations for the female animals was carried out during the gestation and
lactation period.
Additionally, homogeneity and concentration control analyses were carried out at the beginning of the rearing period of the F1 descendant generation.Of each sample one additional reserve sample were take .
Duration of treatment / exposure:
Exposure period: premating (F0 generation) or rearing period (selected F1 animals), mating period, gestation period, lactation period and post
weaning period
Premating exposure period (males): at least 75 days
Premating exposure period (females): at least 75 days
Duration of test: 22 weeks total, the parental animals were exposed to the test substance for ca. 15 weeks
(administration period: 20 Aug 2002 - 05 Jan 2003, animals were sacrificed in Dec. 2002 and Jan. 2003)
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals were not mated.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
nominal in diet
No. of animals per sex per dose:
25 (both F0 and F1)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
100 mg/kg: as the expected "no observed adverse effect level"
300 mg/kg: as intermediate dose level
1,000 mg/kg : as the highest dose level at which possible substance - induced effects but no substance - related mortality in the parental animals
were expected.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once or twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the F0 parental animals was determined on the first day of test substance
administration and then once a week at the same time of the day (in the morning); if possible, the weighings were carried out until the end of the study.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the F0 female animals:
a) During the mating period the F0 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14
and 20 post coitum.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p .p.) and on days 4, 7, 14 and 21 post partum.
d) Females without litter were not weighed during the lactation phase.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all F0 female parental rats for a minimum of 3 weeks prior to mating and were continued
throughout the mating period until the female exhibited evidence of mating. Moreover, at necropsy a vaginal smear was examined to determine the
stage of the estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, sperm head count in right testes and cauda epididymis, sperm motility (all test groups), sperm morphology (only control and high dose group)]
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.


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]


GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after weaning of F1 descendant generation pups the F0 generation parental aninamls were sacrificed]
- Maternal animals: All surviving animals [after weaning of F1 descendant generation pups the F0 generation parental aninamls were sacrificed]


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


HISTOPATHOLOGY / ORGAN WEIGHTS
The following weight parameters of all F0 parental animals sacrificed at scheduled dates were determined:
1. anesthetized parental animals
2. adrenal glands
3. brain
4. epididymides (cauda)
5. epididymides (total)
6. kidneys
7. liver
8. ovaries
9. pituitary gland
10. prostate gland
11. seminal vesicles with coagulating glands (and their fluids)
12. spleen
13. testes
14. thyroid glands (with parathyroid glands)
15. uterus (with oviducts and cervix uteri)

The following organs or tissues of all F0 parental animals sacrificed at scheduled dates were fixed in 4% formaldehyde solution (Fo) or in BOUIN's
solution (B), respectively:
1. adrenal glands (Fo)
2. all gross lesions (Fo)
3. brain (Fo)
4. cervix uteri (Fo)
5. coagulating glands (Fo)
6. kidneys (Fo)
7. left epididymis (B)
8. left testis (B)
9. liver (Fo)
10. ovaries (B)
11. oviducts (Fo)
12. pituitary gland (Fo)
13. prostate gland (Fo)
14. seminal vesicles (Fo)
15. spleen (Fo)
16. thyroid glands, with parathyroid glands (Fo)
17. uterus (Fo)
18. vagina (Fo)
Postmortem examinations (offspring):
SACRIFICE
All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 after birth or subsequent days) were killed by means of CO2. These pups were examined externally and eviscerated; their organs were assessed
macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated, and their organs assessed
macroscopically.
If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals
were, if it was deemed necessary, examined additionally using appropriate methods.

All selected F1 animals were killed by cervical dislocation after sexual maturation (vaginal opening/preputial separation) was determined. These
animals were examined externally and eviscerated; their organs were assessed macroscopically. All animals without any notable findings or
abnormalities were discarded after their macroscopic evaluation.

GROSS NECROPSY
- Gross necropsy consisted of external examinations. (see above)


HISTOPATHOLOGY / ORGAN WEIGTHS
none
Statistics:
statistical evaluations of the clinical examinations were performed using the DUNNETT-test, FISHER'S EXACT test, WILCOXON-test and KRUSKAL-WALLIS test
Reproductive indices:
The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the females were
noted for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = ((number of males with confirmed mating)/(number of males placed with females) x 100)
Male fertility index (%) = ((number of males proving their fertility)/(number of males placed with females) x 100)
The mating partners, the number of mating days until vaginal sperm could be detected and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = ((number of females mated)/(number of females placed with males) x 100)
Female fertility index (%) = ((number of females pregnant)/(number of females mated) x 100)
Gestation index (%) = ((number of females with live pups on the day of birth)/(number of females pregnant) x 100)
Live birth index (%) = ((number of liveborn pups at birth)/(total number of pups born) x 100)
Postimplantation loss (%) = ((number of implantation - number of pups delivered)/(number of implantations) x 100)
Offspring viability indices:
Viability index (%) = ((number of live pups on day 4 after birth)/(number of live pups on the day of birth) x 100)
Lactation index (%) = ((number of live pups on day 21 after birth)/(number of live pups on day 4 after birth) x 100)

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no substance-related mortalities in any of the male and female F0 parental animals in any of the groups.
However, one male F0 animal of test group 01 (100 mg/kg body weight/day) was found dead during study week 7. It showed no clinical findings on the days before its death. Gross and/or histopathological examinations of this male revealed several findings, which may well account for its unscheduled death (e .g. severe embolic-purulent and necrotizing inflammation in the kidneys and a bacterial embolus in the right antrum).
The 3 doses (100; 300 and 1,000 mg/kg body weight/day) administered in the diet did not lead to disturbances of the general behavior in any of the F0 parental animals. Marginal clinical findings, which were considered to be spontaneous in nature, occurred in various animals of different dose groups without any dose-response relationship. One male of the control group had a shortened tail due to a lesion (from study week 7 until terminal sacrifice) and two other males from the same dose group showed skin lesions at their throat from study week 2 respectively 6 until terminal sacrifice.
Malocclusion occurred in one control female (No. 112) from study week 5 until terminal sacrifice. One mid dose female had a palpable mass on the right shoulder from study week 17 onwards and for another female from the same dose group (300 mg/kg body weight/day) hemophthalmia (due to traumatic lesion) on both eyes was observed from study week 9 until terminal sacrifice.
There were no substance-related clinical findings in F0 females during the gestation period for F1 litter.
Other findings, as malocclusion in one control female and hemophthalmia in one mid dose female (No. 165) were already noted during premating and are regarded to be spontaneous in nature.
After the mating period, two sperm positive low dose females and 4 sperm positive mid dose females did not deliver any F1 pups.
The F0 dams showed no test substance-induced clinical findings during the lactation period of the F1 pups.
Malocclusion in one control female was already noted during premating and is, of course, regarded to be spontaneous in nature.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the substance - treated F0 parental males and females was comparable to that of the concurrent controls throughout the entire study. This includes the gestation and the tactation period of the F0 females for the F1 litter. The few statistically significantly increased food consumption values in the F0 parental females of test group 01 (100 mg/kg body weight/day) and test group 02 (300 mg/kg body weight/day) at initiation of the administration period are considered to be without any biological relevance and spontaneous in nature.
Mean body weights and mean body weight gains of the F0 males of test groups 01, 02 and 03 (100, 300 and 1,000 mg/kg body weight/day) were unaffected by the test substance administration during the entire study period and substantially similar to the corresponding control values.
The isolated, however, statistically significantly reduced mean body weight gains of the high dose males (1,000 mg/kg body weight/day) during study weeks 8-9 and 13-14 and the statistically significantly increased body weight gains in mid dose males during study week 12-13 are considered to be spontaneous in nature and without any biological relevance.
Mean body weights and body weight gains of the F0 parental females were not influenced by the test substance administration during premating, gestation, or lactation periods. All differences between the substance-treated females and the control female rats are considered to be incidental and without any toxicological relevance. This includes the statistically significantly higher mean body weight in the low dose females during premating week 4.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The measured intakes of Naphthalene Sulfonic Acids, potassium salts by the different test groups (calculated on the basis of interpolated mean body weights) generally correlated well with the desired target doses.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Evaluation of the estrous cycle data over about 3 treatment weeks prior to mating for the F1 litter revealed generally very regular cycles in the females of all test groups including controls as indicated by a mean cycle duration of 3 .8 (control group), 4 .0 (low and mid dose group), and 4 .2 (high dose group) . The statistically significantly prolonged mean estrous cycle duration at 1,000 mg/kg is not considered as an adverse, substance-induced effect as it is withing the range of the historical controls (3.9 - 5.4 days) and the respective value of the corresponding control group (3 .8 days) is marginally below the lowest value (3.9 days) of several comparable studies.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There occurred no treatment-related changes in the mean number of homogenization resistant testicular spermatids or caudal epididymal sperms, and the percentages of abnormal and normal sperms in the high dose (1,000 mg/kg body weight/day) males.
Sperm motility was not affected by the test substance at any dose level and was substantially similar to the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For nearly all F0 parental males, which were placed with females to generate F1 pups mating was confirmed (indicated by females with vaginal sperm or that gave birth to a litter or with implantation sites/fetuses/pups in utero). The only exception was one control male No. 23 (and its female partner), which both gave no indications for mating. Thus the mating index was 96% in the control group and 100% in the 100, 300 and 1,000 mg/kg group.
For most of the F0 parental males fertility was confirmed within the scheduled mating intervals for F1 litter. The male fertility index varied between 84% and 100% without any relation to dosing.
The following F0 male rats did not prove fertility: one control, two low dose males and four mid dose male rats. However, all high dose males proved their fertility. Gross and histopathological examinations of the 7 males in question failed to show a relevant morphological correlate for the observed impaired fertility. Thus a substance-induced effect on male fertility can be excluded with certainty. The mating index calculated after the mating period for F1 litter was 96% for the control group and 100% for the substance groups indicating that all females (except one control female) were sperm positive or gave birth to a litter or had implantation sites/fetuses/pups in utero.
The mean precoital time (mating until day 0 p.c.) varied between 2.4 and 3 .0 days with no relation to dosing (2.5/3.0/2.4/ and 2.5 days in test groups 00, 01, 02 and 03). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The female fertility index varied between 84% and 100% without showing any relation to dosing. The incidence of non-pregnant animals was one in
the control group, two females in the low dose group, four females in the mid dose group, but none of the high dose females. Gross and histopathological examinations of the 7 females in question failed to show a relevant morphological correlate for the observed impaired fertility. Thus, the differences between the various test groups concerning female fertility indices are not considered to be substance-induced. The mean duration of gestation was very similar in test groups 00-03 (0 ; 100; 300 and 1,000 mg/kg body weight/day) and varied between 21 .8 and 22.0 days with no relation to dosing. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test groups including the controls (10.8 / 12.0 / 11.2 and 10.8 implants/dam in test groups 00 - 03 (0; 100; 300 and 1,000 mg/kg body weight/day)).
Furthermore, there were no indications for substance-induced intrauterine embryo-/fetolethality since the postimplantation loss values were unaffected by treatment.
The mean number of F1 pups delivered per dam was not affected by the test substance administered (10.0 / 11.2 / 10.2 and 9.9 pups/dam at 0 ; 100; 300 and 1,000 mg/kg body weight/day). The number of liveborn and stillborn pups was comparable between the groups, and the live birth index reached 98% (0 mg/kg body weight/day), 99% (300 mg/kg body weight/day) or 100% (100 and 1,000 mg/kg body weight/day), respectively.
Thus, the administration of Naphthalene Sulfonic Acids, potassium salts up to 1,000 mg/kg body weight/day did not adversely affect reproduction and delivery data of the F0 generation parental females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The absolute weights of the seminal vesicles were increased in males by 7.4 % (100 mg/kg bw/day) and 8.1 % (1000 mg/kg bw/day) with p>= 0.05.
The other mean absolute weight parameters of the parental animals did not show significant differences when compared with control group.
The mean relative weight parameters of the parental animals did not show significant differences when compared with the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross lesions were recorded from adrenal cortex (cyst), glandular stomach (erosion/ulcer, heart (focus), kidneys (cyst, focus or pelvic dilation, respectively), liver (focal constriction or focus), mammary gland (cyst), spleen (focus), thyroid glands (organ size reduced) and uterus (focus).
With the exception of "erosion/ulcer" in the mucosa of the glandular stomach and "focus" in the uterus, all these gross lesions occurred only once per group, with no obvious indication of a relationship to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathology failed to correlate the slightly although significantly increased mean absolute weight of the seminal vesicles in the high dose group with a meaningful microscopic finding. This weight increase was regarded to be not related to treatment, as the more reliable mean relative weight was not significantly increased and as no gross lesion or microscopic finding was recorded in any of the animals of this group. The same was true for the slightly although significantly increased mean absolute weight of the seminal vesicles of the low dose group, although histopathology was not performed.
The majority of the other microscopic findings noted in the other organs were either single observations in control or treated animals, or their incidence and graded severity was biologically equally distributed over the dose groups and the control group, giving no indication of a relationship to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects observed in the highest test dose

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index as indicator for pup mortality between days 0-4 p .p. varied between 98% (test groups 00 and 01), 99% (test group 02) and 100% (test group 03), thus showing no compound related changes, but the usual biological variation inherent in the strain of rats used for this study.
The lactation index as indicator for pup mortality between days 4-21 p .p. was not affected by test substance administration in any of the groups.
It reached 99% / 100% / 100% / 99% in test groups 00-03.
Thus, no substance-related differences exist between the control and the 100; 300 and 1,000 mg/kg body weight/day F1 pups concerning viability and mortality.

CLINICAL SIGNS (OFFSPRING)
The F1 generation pups did not show any clinical signs up to day 21, which could be attributed to the treatment.
Only one spontaneous clinical finding (kinked tail), probably due to a traumatic lesion, occurred in one high dose pup (No. 3 from dam No. 177).

BODY WEIGHT (OFFSPRING)
Mean body weights and body weights gains of the F1 pups in test group 01-03 (100; 300 and 1,000 mg/kg body weight/day) were not affected by the test substance administration. The respective values were similar to the corresponding control values and did not show any statistically significant differences. The slight, but statistically significant increase in body weight gain of the low dose (100 mg/kg body weight/day) pups on days 7-14 is a spurious finding, which by itself is not considered to be adverse.

SEXUAL MATURATION (OFFSPRING)
Each selected F1 female animal (reared F1 weanling) was evaluated for vaginal opening.
The first and the last day, when vaginal opening occurred were days 28 and 37 p.p., respectively. The mean number of days to reach the criterion amounted to 30 .8, 32 .2, 32 .2 and 32 .8 days for the 0; 100; 300 and 1,000 mg/kg body weight/day. Vaginal opening occurred in all substance treated groups (100, 300 and 1,000 mg/kg body weight/day) statistically significantly later than in the concurrent control group (1.4 - 2.0 days if expressed on basis of the mean day, when the criterion was reached).
A substance-induced background for the apparent delay in vaginal opening of the selected F1 females seems, however, unlikely for the following reasons:
- the respective value of the corresponding control group (30.8 days) is below the lowest value of several generation studies performed in the same lab under comparable conditions
- 32.2 or 32 .8 days, calculated as mean values for test groups 01 - 03 (100, 300 or 1,000 mg/kg body weight/day), are fully within the range of control values from comparable studies (i.e . 31 .1 - 33.8 days).
Thus, the differences between controls and substance-treated F1 females in the duration until vaginal opening occurred are considered to be spontaneous in nature. The statistically significantly later occurrence of vaginal opening in the substance-treated selected F1 females mirrors a mathematical phenomenon due to the extremely low concurrent control value, which is the lowest "vaginal opening" value recorded in the lab performing the study
until now. A by chance origin applies also for the statistically significantly higher body weights at 100 and 1,000 mg/kg at the time, when the criterion (i .e. vaginal opening) was reached.
Each selected F1 male animal (reared F1 weanling) was evaluated for preputial separation. The first and the last day, when preputial separation occurred were days 40 and 51 p.p., respectively. The mean age for preputial separation was 42.8, 43.4, 42.9, and 43.3 days for the 0; 100; 300 and 1,000 mg/kg body weight/day groups. These values do not suggest any relation to dosing and are fully within the typical biological variatian for the strain of rats used. Thus, preputial separation was not influenced by the treatment.

ORGAN WEIGHTS (OFFSPRING)
There occurred no substance-induced effects on absolute or relative weights of brain, thymus or spleen of the F1 pups. All differences observed reflect the normal biological variation in this strain of rats.

GROSS PATHOLOGY (OFFSPRING)
A few of the large number of F1 pups showed spontaneous findings at gross necropsy or when examined additionally using other appropriate methods. Findings, which occurred, were post mortem autolysis, incisors sloped, hemorrhagic thymus, diaphragmatic hernia, dilated renal pelvis, hemorrhagic testis, and kinked tail. These findings occurred without a clear relation to dosing and/ or most of it can be found in the historical control data at comparable or even higher incidences.
Additionally, skeletal examinations according to a modified method of KIMMEL and TRAMMELL were performed for high dose pup No. 3 of dam No. 177, which showed clinically a kinked tail. The evaluation of the stained skeleton of this pup elicited misshapen and bipartite caudal vertebrae (with changed or split cartilage) and thus confirmed the clinical observation.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects observed in the highest test dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Intake of test substance
The measured intakes of the test substance by the different test groups of the F0 parental animals correlated well with the desired target concentrations:

For the F0 animals during premating:
- between 84.3 - 114.5 mg/kg bw/d in test group 1,
- between 263.6 - 342.5 mg/kg bw/d in test group 2
- between 887.0 - 1395.9 mg/kg bw/d in test group 3.

For the F0 females during gestation:
- 99.5 mg/kg bw/d in test group 1
- 296.1 mg/kg bw/d in test group 2
- 1004.4 mg/kg bw/d in test group 3

For the F0 females during lactation (days 1-14 p.p. only):
- 93.8 mg/kg bw/d in test group 1
- 280.6 mg/kg bw/d in test group 2
- 935.6 mg/kg bw/d in test group 3


F0 parental animal observations
No substance-related mortalities were seen in any of the F0 parental animals in any of the dose groups. One male of the low dose group was found dead during premating (study week 7), without showing clinical findings before its death. Gross and/or histopathological examinations revealed several findings (e.g. severe embolic-purulent and necrotizing inflammation of the kidneys and a bacterial embolus in the right antrum of the heart) that may well account for its unscheduled death.
Marginal clinical findings which were considered to be spontaneous in nature occurred in various animals of different dose groups without any relation to dosing.
For 7 F0 pairs (1/2/4/0 of the control group and test groups 1, 2 and 3) fertility could not be proven because the respective parental F0 females did not deliver any F1 pups. However, sperm evaluation and gross as well as histopathology including differential ovarian follicle count (DOFC) of the male and female non-breeders did not result in convincing findings, which may account for the observed infertility.
With respect to water consumption, there was a statistically significant increase in water consumption in the high dose (1000 mg/kg bw/d) F0 parental animals of both sexes during premating (for male animals for weeks 0-10 about 19% above the corresponding control and for the females about 11% above the corresponding control animals) and for the high dose females during some intervals of the gestation and lactation period (gestation days 0-20 about 24 % and lactation days 1-15 about 14% above the corresponding control values). This increase in water consumption was considered to be substance-induced, but not assessed as an adverse effect as it had to be seen in conjunction with the physico-chemical properties of the test substance, a salt, which had probably made the rats thirstier and led to a marginal increase in water intake.
The isolated, however statistically significant reduction in mean body weight gain of the high dose males during study weeks 8-9 and 13-14 as well as the statistically significant increase in body weight gain in mid dose males during study week 12-13 were considered to be spontaneous in nature and without any biological relevance.
Examination of estrus cycle data revealed very regular cycles in the females of all test groups including the controls. For the control group, a mean cycle duration of 3.8, for the low and mid dose females of 4.0 and for the high dose females of 4.2 days from estrous to estrous were seen. The statistically significantly prolonged mean estrous cycle duration observed at 1000 mg/kg bw/d was not considered as an adverse substance-induced effect. In fact, this mean value predominantly resulted from a single high dose dam that had a mean estrous cycle length of 6.7 days but did not show any other abnormal findings in any other associated parameters. With respect to historical control data the mean estrous cycle length of the females from the substance-treated groups (4.0 - 4.2 days) lies fully within the normal range for rats of the strain used (3.9 - 5.4 days), whereas the control group value (3.8 days) is marginally below the lowest historical control value (3.9 days).
The male mating index was 96% in the control and 100% in the low, mid and high dose groups, respectively. The male fertility index varied between 84 and 100 % without any relation to dosing (i.e. 96% in the control and 92% in the low, 84% in the mid and 100% in the high dose group). The investigation of sperm parameters (i.e. mean number of homogenisation resistant testicular spermatids or caudal epididymal sperms, percentage of abnormal and normal sperms in the high dose (1000 mg/kg bw/d) males and sperm motility) did not show treatment-related changes. Mean sperm motility was 86, 85, 88 and 89% in the control and the low, mid and high dose F0 males, respectively.
The female mating index was 96% in the control and 100% in the low, mid and high dose groups. The mean precoital time (mating until day 0 p.c.) varied between 2.4 and 3.0 days without any relation to dosing (i.e. 2.5/3.0/2.4 and 2.5 days in the control and test groups 1, 2 and 3). The female fertility index varied between 84 and 100% (i.e. 100% in the control, 92% in the low dose, 84% in the mid dose and 100% in the high dose group) without showing a dose-relationship.
The mean duration of gestation varied between 21.8 and 22.0 days in test groups 0-3 without any relation to dosing.
The number of implantation sites was comparable between all test groups including the controls (i.e. 10.8/12.0/11.2 and 10.8 implants/dam in test groups 0-3) and postimplantation loss values were unaffected by treatment. The test substance administration did not affect the mean number of F1 pups delivered per dam and the number of liveborn and stillborn pups was comparable between the groups. The live birth index varied between 98% (control group) and 100% (low and high dose groups).

F1 pups observations:
Regarding pup viability and mortality, the viability index (pup mortality between days 0-4 p.p.) was 98% in control and low dose groups, 99% in the mid and 100% in the high dose group. The lactation index (pup mortality between days 4-21 p.p.) was not affected and was 99% in the control and the high dose groups and 100% in the low and mid dose groups. Thus, no substance-related differences exist between control and test substance-treated groups concerning F1 pups viability and mortality.
Sex distribution and sex ratios of live F1 pups were comparable between controls and treated groups.
Mean body weights and body weight gains of the F1 pups were not affected by the test substance administration and the slight but statistically significant increase in body weight gain of the low dose pups on days 7-14 p.p. is regarded to be a spurious finding and not considered to be adverse.
The F1 pups did not show any clinical signs up to weaning (day 21 p.p.). With respect to necropsy data, a few of the F1 pups showed spontaneous findings at gross necropsy or when examined additionally, i.e. skeletal examination according to a modified method of Kimmel and Trammel (1981) for one high dose pup showing clinically a kinked tail. The findings observed occurred without a clear dose-response relation ship and/or most of them can be seen at comparable or even higher incidences in the historical control data. No substance-induced effects were seen on absolute and relative organ weights of brain, thymus or spleen.

Selected F1 animals observations:
The measured intakes of the test substance by the different test groups of the selected F1 animals from weaning until sexual maturity were as follows:

- for the males and females in test group 1 (100 mg/kg bw/d): 93.3 and 80.9 mg/kg bw/d

- for the males and females in test group 2 (300 mg/kg bw/d): 313.8 and 245.4 mg/kg bw/d

- for the males and females in test group 3 (1000 mg/kg bw/d): 1050.1 and 1261.1 mg/kg bw/d

Neither mortality nor clinical findings were observed the selected F1 groups. Food consumption was unaffected and similar to control values. Mean body weights and body weight gains were unaffected by the test substance administration and comparable to the corresponding control values. This was also true for the isolated but statistically significant increase in mean body weight and body weight gain of the low dose females during study weeks 1-2.

With respect to sexual maturation, the mean age for preputial separation was 42.8, 43.4, 42.9 and 43.3 days for the control group and test groups 1-3. These values were fully within the typical biological variation (42.5-45.0 days) for the strain of rats used and thus, preputial separation was not influenced by the treatment.
Regarding vaginal opening, the mean number of days to reach this criterion amounted to 30.8, 32.2, 32.2 and 32.8 days for the 0, 100, 300 and 1000 mg/kg bw/d groups. Thus, vaginal opening occurred in all substance treated groups statistically significantly later than in the corresponding control group (about 1.5 - 2 days if expressed on basis of the mean day). However, this finding was not regarded to be substance-induced because the value of the control group (30.8 days) was below the lowest value of the historical control data whereas the calculated means of 32.2 and 32.8 days for test groups 1-3 were fully within the range of the historical control data (31.1 - 33.8 days). The authors considered the delay in vaginal opening of the substance-treated F1 females to be spontaneous in nature. Moreover the authors concluded that a by chance origin applies also for the statistically significantly higher body weights at 100 and 1000 mg/kg bw/d at the time, the criterion was reached.
At necropsy there were only 4 of a total of 200 selected F1 rats that showed spontaneous findings in the form of hemorrhagic thymus (in 2 mid dose males) and dilated renal pelvis (in two high dose males from the same litter). Due to the scattered occurrence of these findings and due to the reason that these findings also occurred in control F1 pups and were present in the pup historical control data at comparable or even higher incidences, these findings were not considered to be substance-induced.

Clinical pathology of the F0 parental animals:
The only test substance-related effects occurred in the high and the mid dose groups (1000 and 300 mg/kg bw/d) in the form of decreased total bilirubin concentrations in both sexes (in test group 3 -24% in the males and -33% in the females, in test group 2 -13% in the males and -22% in the females). However, the slight decrease in total bilirubin had no pathognomonic relevance and was considered not to be toxicologically significant.
No test substance-related effects were seen in haematological parameters and in urinalyses.

Organ weight parameters of the F0 parental animals:
The only statistically significant finding was an increase in absolute weight of the seminal vesicles of the males in test group 1 (7.4%) and test group 3 (8.2%). However, no histopathological correlate could be found in test group 3 (histopathology of seminal vesicles was not performed in the low dose group) and the more reliable mean relative weight was not significantly increased, thus this finding was not regarded to be substance-related.

Gross lesions:
The gross lesions observed (cyst in adrenal cortex, erosion/ulcer in glandular stomach, focus in the heart, cyst, focus or pelvic dilation in kidneys, focal constriction or focus in the liver, cyst in mammary gland, focus in the spleen, organ size of thyroid glands reduced, focus in the uterus) occurred with the exception of two findings (erosion/ulcer in the glandular stomach and focus in the uterus) only once per group and without an obvious indication of a relationship to treatment.
The examination of the 7 females, which were not pregnant (1/ 2/ 4/ 0 in the control, low, mid and high dose groups), together with their mating partners did not reveal gross lesions that accounted for the animals' status "animal not pregnant".

The results of Differential ovarian follicle count (DOFC) did not reveal significant deviations between the animals of control group and the animals of the high dose group. There was no indication that the test substance administration had negatively influenced morphology and/or function of the ovaries, oviducts, uterus, cervix uteri or vagina as well as of the testes, epididymides (including cauda, epididymidis), prostate gland, seminal vesicles or coagulations glands of parental animals of the F0 generation.

According to the authors, the test substance had no adverse effects on fertility and reproductive performance of the F0 parental animals at the doses of 100, 300 and 1000 mg/kg bw/d.
Clinical pathology examinations of the parental animals revealed only slightly decreased bilirubin concentrations in the serum of the mid and high dose animals of both sexes. These isolated findings were probably related to the test substance administration. However, in general, i.e. in the absence of any other sign of systemic toxicity, in particular of clinical pathology and pathological alterations, the observed decrease in serum bilirubin concentration has no pathognomonic or toxicological relevance.
With respect to the F1 pups and the selected F1 animals (reared F1 weanlings) no test substance-related adverse effects were seen in any of the dose groups regarding clinical examinations, pup organ weights and sexual maturation.
No substance-induced signs of developmental toxicity in the progeny of the F0 parents were found.
Thus, the authors concluded that under the conditions of the present extended one-generation reproduction toxicity study the no observed adverse effect level (NOAEL) of 1000 mg/kg bw/d could be fixed for
- fertility and reproductive performance in both sexes of the F0 parental rats
- overall general toxicity of the test substance
- and for developmental toxicity (i.e. growth and development of the F1 offspring until sexual maturity).

 

Applicant's summary and conclusion

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