Cyclohexane, 1,4-bis(ethoxymethyl)-

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Experimental study according to guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1983

Principles of method if other than guideline:

In addition the study was extended by parameters which were part of the following test guidelines:
- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Two-Generation Reproduction Toxicity Study; Official Journal of the Union, No. L 142, pp. 355-364
- EPA Health Effects Test Guidelines, OPPTS 870.3800: Reproduction and Fertility Effects (Aug 1998)
- OECD Guidelines for Testing of Chemicals; No. 416 (22 Jan 2001)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

EC Number:

700-868-7

Cas Number:

54889-63-3

Molecular formula:

C12 H24 O2

IUPAC Name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

Details on test material:

- Name of test material: 1,4-bis(ethoxymethyl)-Cyclohexane 97 %
- Physical state: Liquid/colorless, clear
- Storage: Room temperature
- Expiry date: 2016-03

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: F0 (parents): 32 ±1 days; F1 (pups):
- Weight at study initiation: (F0) Males: 89.3 g - 124.6 g; Females: 86.7 g - 105.4 g
- Fasting period before study: no
- Housing: During premating and postmating animals were group-housed in Polysulfonate cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany. During mating, gestation and lactation the rats were housed individually in Polycarbonate cages type III supplied by Becker & Co., Castrop-Rauxel, Germany. During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 21. For enrichment wooden gnawing blocks and play tunnels were provided.
- Diet: ad libitum, Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in corn oil were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with corn oil and subsequently thoroughly mixed with a magnetic stirrer until it was completely dissolved. During administraton the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: The test substance was completely miscible with corn oil and stable in corn oil.
- Concentration in vehicle: 0.125, 0.375, 1.250 mg/100 mL
- Amount of vehicle: 4 mL/kg bw /day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear (referred to as day 0 of pregnancy)
- Further matings after unsuccessful attempts: No, each female animal was paired with a predetermined male animal from the same dose group.
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 21.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in corn oil for a period of 7 days at room temperature were carried out before the study was initiated. Given that the test substance was completely miscible with corn oil, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory three times during the study period for verification of the concentrations.

Duration of treatment / exposure:

At least 69 days before mating and continued up to one day prior to sacrifice. Females in labor were not treated. Female animals were sacrifized 123 or 126 days after the treatment start. Male animals were sacrifized after 100 or 105 days of treatment.

Frequency of treatment:

daily

Details on study schedule:

After the acclimatization period, the test substance was administered to the parental animals. F0 parental animals not mated until 69 days after selected from the F0 litters. The mating was continued for up to two weeks. Females in labor were not treated with the test substance. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21. After weaning of F1 pups the F0 generation parental animals were sacrificed. Blood samples were taken of the F0 parental animals shortly before sacrifice. Due to the differences in mating duration the parental generation was not sacrifized on one day. While male animals were sacrifized after 100 or 105 days of treatment, the female animals were sacrifized 123 or 126 days after the first administration. All pups were sacrificed under isoflurane anesthesia with CO2 after standardization (PND 4) or weaning (PND 21).

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15, 50 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In order to obtain information on the effect of the test substance Cyclohexane, 1,4-bis(ethoxymethyl)- on male sexual organs after repeated oral administration (gavage) a Screening Study on Testes Toxicity in Male Wistar Rats was (BASF SE, 2015, 00R0804/11N025) was performed. The test substances were administered at dosis of 1000 mg/kg bw once per day to groups of 5 male Wistar rats via gavage for a maximum of 14 days. During the study, all animals were observed for any clinically abnormal signs. One day after the last administration the animals were sacrificed and assessed by gross pathology, special attention was given to the reproductive organs. A decreased food consumption (-33%) and body weight loss (-13%) were observed. No test substance-related relevant findings were noted concerning sperm parameters. The following effects were noted on male sexual organs: decreased absolute weights (i.e. -10%) of epididymides, decreased absolute weights (i.e. -35%) of seminal vesicles, decreased absolute weights (i.e. -12%; not statistically significant) of testes and degeneration of pachytene spermatocytes with single multinucleated cells in 5/5 animals in the left testicle. Because of severe body weight loss the study cannot be used for assessment, as the observed effects may be secondary to the treatment and not substance specific.
To avoid secondary effects resulting from peak exposure a feeding study with Cyclohexane, 1,4-bis(ethoxymethyl)- in male Wistar rats was perfomed (BASF SE, 2014, 00R0652/13N073). The encapsulated test substance (w: 46.65% 1,4-bis(ethoxymethyl)-Cyclohexane and w: 46.57% corn oil) was administered in concentrations of 6000 and 12000 ppm (active ingredient) via the diet for 14 days. Because of the continuing body weight loss (19% in the low dose group and 14% in the high dose group from study day 0 to 10), the reduced food consumption (down to 67% in the low-dose and high-dose group in comparison to the control) and the poor general state of all test substance treated animals, e.g. piloerection and hunched posture, these animals were sacrificed moribund. The vehicle control groups have been sacrificed unscheduled for reasons of humanity. The study was terminated before the experimental completion date and cannot be used therefore for assessment.
A range finder study was performed to select the appropriate dose levels for a One-Generation Reproduction Toxicity Study in Wistar Rats. Therefore, 5 male and female Wistar rats/group were dosed with 200 and 600 mg/kg bw/day. As food consumption was reduced and a body weight loss in male and female animals was observed, the animals were scarified on study day 25. Another 28-day range finder study was performed administering 10 and 50 mg/kg bw/day to investigate toxicological effects at or below the maximal tolerable dose. No test substance-related findings with regard to body weight, hematology, clinical chemistry in males and females were observed. Sperm analysis did not show any abnormalities. The histopathological investigation of the testis revealed a minimal tubular degeneration and a minimal luminal debris in the corresponding epididymidal tubules in 2 (out of 5) animals. In order to obtain general information on the possible effects of 1,4-bis(ethoxymethyl)-Cyclohexane on the integrity and performance of the male and female reproductive systems including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation and weaning, as well as on growth and development of offspring from one generation of Wistar rats a modified One-Generation Reproduction Toxicity Study was performed with dose levels of 5, 15 and 50 mg/kg bw/day (BASF SE, 2016, 70R0261/14R090).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Starting from study day 4 onwards the examination was conducted before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal.

DETAILED CLINICAL OBSERVATIONS:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT: Yes
- Time schedule for examinations: once per week.
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- During lactation, body weight of the F0 females, which gave birth to a litter was determined for PND 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Generally, food consumption was determined once a week for male and female F0 parental animals, with the following exceptions:
-Food consumption was not determined after the 10. premating week (male F0 animals) and during the mating period (male and female F0 animals).
-During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0 - 7, 7 - 14 and 14 - 20.
-During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1 - 4, 4 - 7, 7 - 14, and 14 - 21.
Food consumption was not determined for F0 females without positive evidence of sperm and females without litter.

OTHER: Haematology (see table 1) and Clinical chemistry (see table 2).

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. 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):

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males at scheduled sacrifice or after appropriate staining. The left testis and left epididymis of all animals sacrificed at scheduled dates was fixed in modified Davidson’s solution, whereas the right testis and epididymis was used to determine sperm parameters.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 4 pups/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, Nipple/areola anlagen, Anogenital index, organ weight

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
Shortly before weaning of the F1 pups the F0 generation parental male animals were sacrificed.
After weaning of F1 pups the F0 generation parental female animals were sacrificed.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 and 4 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

All pups with scheduled sacrifice (i.e. pups culled on PND 4 or sacrificed on PND 21) were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

Please refer to table 5 in "any other information on materials and methods".

Reproductive indices:

For male and female animals and pups following indeces (in %, except sex ratio, anogenital index) were calculated:
- Male: mating index, fertility index
- Female: mating index, fertility index, gestation index, live birth index, postimplantation loss
- Pups: viability index, lactation index, sex ratio, anogenital index

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4, 5 - 7, 8 - 14 and 15 - 21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Test substance intake: The test substance was administered via gavage therefore no substance intake was monitored. General food consumption was evaluated.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Several animals showed salivation after treatment. In addition one female animal of test group 3 (50 mg/kg bw/day) showed blood in bedding on GD 23. Two sperm positive females of test group 3, one sperm positive female of test group 2 (15 mg/kg bw/day) did not deliver F1 pups. One female of test group 3 (50 mg/kg bw/day) and one female of test group 0 (control) had a complete litter loss on PND 0.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test substance-related changes of food consumption were noted in males of all dose groups during the entire study. The statistically significantly increased food consumption in the high-dose males during premating days 14 - 21 was considered to be spontaneous in nature. No test substance-related changes of food consumption were noted in females of all dose groups during premating and gestation. In the low- and mid-dose groups (5 and 15 mg/kg bw/day) no change of food consumption was observed during lactation. Slightly lower food consumption in all test substance-treated groups at the beginning of gestation was not consistent and thus not considered to be treatment-related. Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during PND 1 - 14 and when summarized for the entire lactation (PND 1 – 21; up to 24 % and 15 %, respectively).
Mean body weights and body weight gain of all test substance-treated F0 male animals were comparable to the concurrent control values throughout the entire study. Statistically significant decreases or increases of body weight gain scattered across the study period (premating days 14 – 21, 56 - 63 and postmating days 7 – 14) were considered to be spontaneous in nature. The mean body weights of the high-dose parental females (50 mg/kg bw/day) were statistically significantly below the concurrent control values on GD 20 (about 7 %) and during PND 4 – 14 (up to 8 %). Statistically significantly lower mean body weights were also noted for the mid-dose parental females (15 mg/kg bw/day) during gestation and lactation. Although not always dose-related (like on GD 7 and 14, and on PND 0) the overall change is suggestive of a treatment-related effect. In contrast the slightly lower weight on premating day 63 is most likely an incidental finding. No test substance-related changes of mean body weights were observed in the low-dose group (5 mg/kg bw/day) during the entire study. Body weight gain values of test substance-treated females gave no uniform picture across all dose groups. Whereas there was no change at all noted during premating, a decreased weight gain was observed in females of all treated groups towards the end of gestation. On the other hand weight gain of the treated females was overall higher than control during lactation although the difference was not statistically significant. Specifically in the high-dose group a decreased weight gain at the beginning of lactation (PND 0-4) was followed by an increased weight gain during the remaining lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: approx. 4 days in all test groups.

REPRODUCTIVE FUNCTION: MALE (PARENTAL ANIMALS)
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two high-dose males, one mid-dose male and one control male did not generate pregnancy in their female mating partners. One male of test group 3 (50 mg/kg bw/day) generated pregnancy (implants in the uterus) of its female mating partner, but the female did not deliver F1 pups. Thus, the male fertility index ranged between 92 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross or microscopic lesion lesions, except for one control male which showed a severe tubular degeneration in the left testis and aspermia, accompanied by severe cell debris and focal cribiform change in the left epididymis.

REPRODUCTIVE FUNCTION: MALE SPERM PARAMETERS (PARENTAL ANIMALS)
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

REPRODUCTIVE FUNCTION: FEMALE (PARENTAL ANIMALS)
The female mating index calculated after the mating period for F1 litter was 100 % in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.2 days without any relation to dosing. Most female rats delivered pups or had implants in utero. Only 2 females from test group 3 (50 mg/kg bw/day), 1 from test group 2 (15 mg/kg bw/day) and one control female did not become pregnant. The fertility index varied between 92 % (test group 3), 96 % (test group 2 and control) and 100 % (test group 1). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross or microscopic lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.2 and 22.3 days). The gestation index was 95.7 % in the high-dose group, 95.8 % in control and 100 % in the mid- and low-dose group. The mean numbers of implantation sites were 12.0 / 10.3** / 11.0 and 9.2** implants/dam in test groups 0 - 3, respectively. The numbers were statistically significantly lower in test groups 1 and 3, but slightly outside the historical control range (9.4 – 14.0) in test group 3 only. The post-implantation loss did not show any significant differences between the treated groups and the control. The mean numbers of F1 pups delivered per dam was statistically significantly lower in test groups 1 - 3 (11.2 / 9.8* / 10.2* and 9.4* pups/dam in test groups 0 - 3, respectively). The mean number of F1 liveborn pups delivered per dam was statistically significantly lower in test groups 1 - 3 (10.8 / 9.7* / 10.2* and 9.3* pups/dam in test groups 0 - 3, respectively). However, the number of stillborn pups was not significantly different between the test groups and the concurrent control.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In male animals, the significant decrease of the absolute and relative weight of the seminal vesicles was not dose-dependent and the values were within the historical control range (absolute: 1.041 – 1.327g; relative: 0.273 – 0.332 %). The significant relative weight increase of the kidneys (0.642 %) was within the normal historical control range (0.577 – 0.671 %). Therefore, all of these significant organ weight deviations in male animals were considered to be not treatment-related. In the spleen, only the absolute weights were decreased in test groups 2 and 3 but no changes were noted in the relative weights. Although no histopathological evaluation of the spleen was performed, the significant absolute weight decreases were considered incidental and not related to treatment.
In female animals, the significant absolute weight increase of the adrenal glands (absolute: 78.435 mg) was within the historical control range (67.000 – 85.700 mg). Although the relative weight increase (0.036 %) was minimally above the historical control range (0.031 – 0.035 %), no histopathological correlate was found. Therefore, the weight increases are considered to be incidental and not related to treatment. The significant decrease of the absolute weight of the ovaries (95.913 mg) was below the historical control range (98.400 – 125.200 mg), whereas the relative weight decrease (0.044 %) was within the historical control values (0.041 – 0.051%). Since no histopathological correlate was seen in the ovaries of females that can explain the minimal weight deviations, the weight decrease is considered incidental and not related to treatment. The significant relative weight increase of the liver (3.1 %) was within the historical control range (2.464 – 3.347 %) and was therefore judged as not treatment-related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Compared to the control group, the minimal increase in the incidence of foci in the glandular stomach of test group 3 female animals was regarded as possible treatment-related and correlated with histopathological erosions/ulcers. All other 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.
Deceased animals
A red discoloration and foamy content was found in the lungs of 2 female animals which died intercurrently.
Fertility
The female animals which were not pregnant as well as the male mating partners showed no relevant gross lesions. Only one control male showed reduced size of the epididymides and testes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Compared with controls, there was an increased incidence of tubular degeneration (9 out of 25 animals) in the left testis of rats dosed with 50 mg/kg bw/dayay. The tubular degeneration was characterized by occasional tubules that were partially depleted of germ cells. The germ cell depletion sometimes affected only a focal region within a tubular cross section. The severity of the finding was minimal in all cases, affecting only a few or a small proportion (<5 %) of tubular cross sections. Two rats in the control group also had tubular degeneration, but the germ cell depletion had a different distribution pattern to that seen in the treated rats. In one control rat graded severe, the germ cell depletion diffusely affected all tubules and was associated with aspermia and cell debris in the epididymis. In the other control (graded slight), a group of adjacent tubular cross sections were diffusely depleted of germ cells. This contrasted with the focal or partial germ cell depletion in occasional tubules (graded minimal) in the 50 mg/kg bw/dayay rats. The tubular degeneration in the two controls and in a single rat dosed with 5 mg/kg bw/dayay were considered incidental findings and were similar to those described as background lesions in control rat testes. The increased incidence of minimal tubular degeneration in the testes of the 50 mg/kg bw/dayay group was considered test substance related. However, it was not associated with any changes in testis weight and was not accompanied by any findings in the epididymis or any changes in sperm parameters (count, motility or morphology). Based on the relatively low incidence (9/25) and the minimal, focal nature of the germ cell loss, plus the absence of any functional or quantitative effects on sperm parameters, the finding of tubular degeneration in the 50 mg/kg/day group is considered non adverse. Minimal erosion/ulceration found in the glandular stomach of 3 out of 25 females of test group 3 (50 mg/kg bw/day) was regarded as possible treatment-related but was not considered adverse. All other findings occurred either individually or were biologically equally distributed about control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Deceased animals
The lungs of two animals showed slight congestion accompanied by a slight alveolar edema and alveolar histiocytosis. These findings most likely represent a lung edema and correlated with the macroscopic changes.
Fertility
The female animals which were not pregnant as well as the male mating partners showed no relevant histopathological findings. Only one control male showed severe tubular degeneration in the left testis and aspermia, accompanied by severe cell debris and focal cribiform change in the left epididymis. These findings correlated with reduced size of both organs.

OTHER FINDINGS (PARENTAL ANIMALS)
No treatment-related changes among hematological parameters were observed.
In females of test groups 2 and 3 (15 and 50 mg/kg bw/d) mean corpuscular volume (MCV) was higher compared to controls. MCV is a calculated index and the measured red blood cell parameters (i.e., red blood cell (RBC) counts, hematocrit and hemoglobin values) were not altered. Therefore, the MCV change was regarded as incidental and not treatment-related.

No treatment-related changes among clinical chemistry parameters were observed.
In males of test group 3 (50 mg/kg bw/d) total protein, albumin, globulin and calcium levels were decreased and inorganic phosphate levels were increased. Total protein means were slightly above the historical control range, but the two protein fractions, albumin and globulins, were within historical control ranges (total protein 60.96-65.38 g/L; albumin 34.13-41.09 g/L; globulins 22.49-28.23 g/L). Total protein mean in males of test group 3 was only 3 % lower than that of the study control. Calcium and inorganic phosphate values were also within historical control ranges (calcium 2.45-2.67 mmol/L; inorganic phosphate 1.36-1.82 mmol/L). In males of test group 2 (15 mg/kg bw/d) creatinine values were lower compared to controls, but the change was not dose-dependent. Therefore, all mentioned parameters including the altered total protein values in males of test group 3 (50 mg/kg bw/d) and in males of test group 2 (15 mg/kg bw/d) were regarded as incidental and not treatment-related.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on reduced food consumption and/or reduced body weight gain towards the end of gestation and 14 days into lactation

2

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed in the highest dose group.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 99.0 % / 98.1 % / 99.3 % and 95.1 % in test groups 0 - 3. The lactation index indicating pup mortality during later lactation (PND 4 - 21) varied between 95.7 % / 100 % / 100 % and 90.5 % in test groups 0 - 3. The apparently lower percentage in the high-dose group was caused by two litters the offspring of which had to be sacrificed after the accidental death of their mothers.

CLINICAL SIGNS (OFFSPRING)
The F1 generation pups did not display any clinical signs until weaning.

BODY WEIGHT (OFFSPRING)
Mean body weights of the male and female pups of test groups 3 were statistically significantly below the concurrent control values during PND 7 - 21 (between 11 and 17 % below control), as was pup body weight gain from PND 4 onwards. No test substance-related influence on body weights and body weight change of F1 pups were noted in the low- and mid-dose groups. The statistically significantly higher pup body weights and pup body weight change in the low dose group during several parts of the lactation period were considered to be spontaneous in nature.

SEXUAL MATURATION (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

ORGAN WEIGHTS (OFFSPRING)
The decreased absolute spleen and thymus weights, decreased relative spleen weights and increased relative brain weights in the high-dose F1 pups were considered to be secondary to the lower pup body weights in these groups. All other pup organ weight changes were not related to dose and thus considered of no toxicological relevance.

GROSS PATHOLOGY (OFFSPRING)
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, hydroureter, diaphragmatic hernia, discolored thymus (reddish), dilated renal pelvis, hydronephrosis, discolored liver lobe (white) and incisors sloped. 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.

OTHER FINDINGS (OFFSPRING)
The mean number of F1 pups delivered per dam was statistically significantly decreased in test groups 1 - 3 (11.2 / 9.8* [*=p<=0.05] / 10.2* and 9.4* pups/dam in test groups 0 - 3, respectively). The mean number of liveborn pups was statistically significantly decreased in test groups 1 - 3 (10.8 / 9.7* [*=p<=0.05] / 10.2* and 9.3* pups/dam in test groups 0 - 3, respectively). The number of litters with stillborn and cannibalized pups was evenly distributed about the test groups.

The number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. Owing to the high background rate, the statistically significantly elevated percentage of nipple in the high-dose males on PND 13 was considered to be an incidental finding. No areolae at all were detected during re-examination one day prior to pup necropsy (PND 21).

No test substance-related effects on anogenital distance or anogenital index were noted in all treated F1 offspring (test groups 1 - 3 [5; 15 and 50 mg/kg bw/d]). The statistically significantly increased anogenital distance in male pups of the low-dose group was considered to be an incidental finding.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on slightly decreased pre-weaning pup body weights/pup weight gain, secondary to decreased food consumption/body weight gain in the corresponding F0 parental females

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Absolute organ weights

When compared to control group 0 (set to 100 %), the mean absolute weights of following organs were significantly changed:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (5)	2 (15)	3 (50)	1 (5)	2 (15)	3 (50)
Seminal vesicles	94%	86%**	91%*
Spleen	101%	91%*	89%**
Adrenal glands				93%*	99%	109%*
Ovaries				98%	93%	86%**

*p ≤ 0.05; **p ≤ 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

 

Relative organ weight

When compared to control group 0 (set to 100 %), the mean relative weights of following organs were significantly changed:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (5)	2 (15)	3 (50)	1 (5)	2 (15)	3 (50)
Kidneys	98%	101%	105%*
Seminal vesicles	91%	88%**	94%
Adrenal glands				95%	103%	112%**
Liver				100%	104%	108%**
Ovaries				101%	97%	88%*

*p ≤ 0.05; **p ≤ 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

 

Gross lesions

In the glandular stomach of females, red or black foci were seen with following incidence:

	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (5)	2 (15)	3 (50)
No. of animals	25	25	25	25
Focus	1	1	2	3

 

Histopathology

Treatment-related findings were observed in the left testis with incidences and grading according to the table below:

	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (5)	2 (15)	3 (50)
No. of animals	25	25	25	25
Degeneration, tubular	2	1	0	9*
Grade 1		1		9
Grade 2	1
Grade 4	1

* : P <= 0.05, **: p <= 0.01

 

 

Pup organ weight

Absolute pup organ weights, compared to control (= 100 %)

F1 pups	Test group 1 (5 mg/kg bw/day)	Test group 2 (15 mg/kg bw/day)	Test group 3 (50 mg/kg bw/day)
Brain (female)	102%*	102%*	98%
Brain (male+female)	102%*	102%*	98%
Spleen (male)	106%	102%	79%**
Spleen (female)	93%	99%	70%**
Spleen (male+female)	99%	100%	74%**
Thymus (male)	108%*	108%	84%*
Thymus (female)	100%	99%	81%**
Thymus (male+female)	104%	103%	83%**

* or ** = p-level of significance ≤ 0.05 or ≤ 0.01, respectively

 

Relative pup organ weights, compared to control (= 100%)

F1 pups	Test group 1 (5 mg/kg bw/day)	Test group 2 (15 mg/kg bw/day)	Test group 3 (50 mg/kg bw/day)
Brain (male)	98%	100%	111%**
Brain (female)	102%	102%	114%**
Brain (male+female)	100%	101%	113%**
Spleen (male)	103%	100%	88%*
Spleen (female)	93%	100%	81%**
Spleen (male+female)	98%	100%	85%**
Thymus (male)	105%	107%*	95%

* or ** = p-level of significance ≤ 0.05 or ≤ 0.01, respectively