Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25.10.2012 - 08.01.2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD 422 guideline study under GLP, but problems with light / dark cycle during study resulting in effects reducing reliability of the study; therefore a Klimisch rating of 2 was applied.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Body weights of animals at the start of treatment were slightly higher than study plan. Automatic light cycle of 12 h on / 12 h off was not functional --> constant lighting during the conduct of this study --> increased number of non pregnant females.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear liquid
Details on test material:
Colorless to pale yellow, clear liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 50 males: 10 per group, 50 females: 10 per group
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males: 312 to 396 g, Females: 212 to 254 g
Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

In several studies conducted in November/December 2012 increased number of non pregnant females was noted. It was noted on 01-Feb-2013, that the automatic light cycle of 12 h on / 12 h off was not functional within a block of rooms including room number 11A. Therefore it was concluded that a constant lighting during the conduct of this study was present. Because of pre-termination of two females in group 5 during the pre-pairing period, two males were not paired during the first pairing period. These males were used for the second pairing period instead of males which already mated successfully.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Study Sequence Females Males
Acclimatization 5 days minimum 5 days minimum
First Test Item Administration Day 1 of pre-pairing Day 1 of pre-pairing
Pre-Pairing 14 days 14 days
Blood Sampling Day 14 of pre-pairing Day 14 of pre-pairing
Pairing 14 days maximum 14 days maximum
Gestation Approximately 21 days
Treatment Ends On day 3 post partum On day before sacrifice
Necropsy Dams and pups on day 4 post partum After treatment for 47 days,
when no longer needed for assessment of reproductive effects
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to B. Bürkle (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Duration of treatment / exposure:
Target Dose Levels:
Group 1: 0 mg/kg/day (control group)
Group 2: 60 mg/kg/day
Group 3: 120 mg/kg/day
Group 4: 300 mg/kg/day
Group 5: 750 mg/kg/day
Dose Volume: 4 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL
Group 2: 15 mg/mL
Group 3: 30 mg/mL
Group 4: 75 mg/mL
Group 5: 187.5 mg/mL
Frequency of treatment:
Frequency of Administration: Once daily
Duration of test:
Duration of Treatment Period:
Males: 47 days
Females: Approximately 7 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 120, 300, 750 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent no treatment

Examinations

Maternal examinations:
The following observations were recorded:
Viability / Mortality: Twice daily
Clinical Signs:
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption:
Males: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; after pairing period weekly.
Females: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; gestation days 0 - 7, 7 14 and 14 - 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.
Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possi¬ble), 1 and 4 post partum.
Ovaries and uterine content:
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Fetal examinations:
Males were sacrificed after treatment after treatment for 47 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 4 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All parent animals and pups, except those excessively cannibalized, were examined macroscopi-cally for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
For the parent animals, special attention was directed at the organs of the reproductive system.
Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: The NOAEL for materna toxic effects was established at 300 mg/kg bw/d

Details on maternal toxic effects:
Reduced food consumption, reduced body weights and 2 deaths occurred at 750 mg/kg bw/day. Increased liver and kidney weights occurred at 300 mg/kg bw/day. Mild centrilobular hepatocyte hypertrophy and diffused thyroid follicular epithelium hypertrophy was observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
other: no effect on development
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: equivocal effects at 750 (mg/kg bw/d) at levels of maternal toxicity

Details on embryotoxic / teratogenic effects:
At the dose level of 750 mg/kg bw/day, number of corpora lutea in female nos. 93, 94 and 95 was 8, 9 and 10, respectively, compared to the mean number of corpora lutea of 13.5 in the control group. Due to the low number of pregnant females these results are equivocal. However, because corpora lutea count was lower in all three females compared to the control mean value, a test item-related effect should be taken into consideration.
No effects on corpora lutea count were observed up to the dose level of 300 mg/kg bw/day.
Mean number of corpora lutea per dam was 13.5, 13.8, 16.0 and 13.9 at the dose levels of 0, 60, 120 and 300, respectively

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(60)

3
(120)

4
(300)

5
(750)

Female numbers

51 - 60

61 - 70

71 - 80

81 - 90

91 – 100

Number of females paired (A)

10

10

10

10

8

Number of females mated (B)

10

10

10

10

7

Number of females not pregnant (C)

2

6

5

3

4

Number of pregnant females which did not give birth to living pups (D)

1

0

0

0

2

Number of females with living pups at first litter check

7

4

5

7

1

Number of females which lost their litters (E)

0

0

0

0

1

Number of females which reared their pups until day 4post partum

7

4

5

7

0

(A)   Female nos. 92 and 97 were terminated during pre-pairing period,

(B)   Female no. 91 did not mate,

(C)   Female nos. 52, 58, 63, 64, 65, 68, 69, 70, 71, 72, 74, 76, 77, 82, 87, 90, 96, 98, 99 and 100,

(D)   Female no. 57 had only one dead pup at first litter check, no. 93 had fetuses at termination on day 25p. cand female no 94 had only implantation sites at termination on day 25p. c.,

(E)   Female no. 95 missed its single pup on day 2p. p.

Applicant's summary and conclusion

Conclusions:
Male-rat specific hydrocarbon nephropathy was observed at all doses tested, but not considered relevant for human risk assessment. Adaptive liver changes occurred as weight increase and minimal or mild centrilobular hepatocyte hypertrophy, consistent in appearance with enzyme induction, in males at 120 mg/kg bw/day and higher and in females at 300 mg/kg bw/day and higher. Secondary to liver enzyme induction, a greater incidence and/or severity of diffuse hypertrophy of the follicular epithelium of thyroids was noted in females at 300 mg/kg bw/day and in both sexes at 750 mg/kg bw/day. Based on adverse effects on food consumption and body weights in both sexes and mortality of females noted at the dose level of 750 mg/kg bw/day, a NOAEL for general toxicity was established at 300 mg/kg bw/day for both sexes.
Reproduction was affected down to the dose level of 300 mg/kg bw/day, where increased post implantation loss was noted. Under the conditions of this study, no indication of any effect on reproduction was noted at the dose levels of 60 and 120 mg/kg bw/day. However, because of low numbers of pregnant females, none of these dose levels could be conclusively confirmed as NOAEL.
Executive summary:

This study is an investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Homosalate to rats. Homosalate was administered in corn oil as vehicle at dosages of 60, 120, 300, and 750 mg/kg bw/day to male rats for 47 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3post partum, one day before necropsy. Control animals received the vehicle only. Due to an technical error, animals were exposed to a constant lighting instead of 12 hours light/12 hours dark cycle during the conduct of this study. As consequence, increased infertility was observed in all groups. This effect was distributed in a dose level independent manner: number of pregnancies in the groups 2, 3 and 5 were very low whereas the number in the control group and group 4 was similar to the normal background values (8 and 7, respectively). The low number of pregnancies per group might have had an impact on evaluation of data on breeding and reproduction at the dose levels of 60, 120 and 750 mg /kg bw/day. Eight pregnancies in the control group and seven pregnancies at the dose level of 300 mg/kg bw/day enabled reliable evaluation of the data. General Toxicity Under the conditions of this study, test item-related adverse effects of general toxicity were noted at the dose level of 750 mg/kg bw/day.  Two females at the dose level of 750 mg/kg bw/day were terminated prior to schedule: one female was found dead and one further was killed for ethical reasons on day 7 of the pre-pairing period. Body weight loss and clinical signs, indicating bad condition were noted in these females. All remaining animals had no adverse clinical signs and survived the scheduled study period. At the dose level of 750 mg/kg bw/day, reduced food consumption, reduced absolute body weights and reduced body weight gain were noted in males and females. Food consumption was affected in both sexes during the pre-pairing period, but recovered afterwards. Reduction in body weights was noted in males starting from day 3 of the pre-pairing until completion of the study whereas significant reduction in body weight gain was noted during the pre-pairing period and at the end of the after pairing period. In females, body weight loss was noted during the first 7 days of the pre-pairing and reduced body weight gain until the completion of this period resulting in reduction in absolute body weights noted during the entire pre-pairing period. During gestation, body weight gain of female no. 95 was initially similar to the control value and lower than the mean control values starting from day 13 of this period onwards whereas absolute body weight was lower than the mean body weights of females in the control group. Effects on food consumption and body weights were considered to be adverse. No effects on food consumption or body weights were noted up to the dose level of 300 mg/kg bw/day. During clinical laboratory investigations, higher concentration of albumin and lower concentration of globulin resulting in higher albumin / globulin ratio were noted in males at the dose level of 750 mg/kg bw/day. Higher albumin concentration was noted in males at the dose level of 300 mg/kg bw/day. These changes were considered to probably be related to the treatment with the test item. No further test item-related changes in hematology or biochemistry parameters were noted in males or females at any dose level. During necropsy, at the dose level of 750 mg/kg bw/day, increased liver weights and reduced thymus weights were noted in both sexes as well as increased kidney weights were noted in females. Further, weights of prostate and seminal vesicles were reduced in males at this dose level. At the dose level of 300 mg/kg bw/day, increased liver weights were noted in both sexes and increased kidney weights were noted in females. Reduction in prostate and seminal vesicle weights was considered to be test item-related adverse effect. Test item related findings noted during histopathological examination, were considered not to be adverse. In kidneys, hyaline droplet nephropathy in males from all the treated groups as evidenced by a minimal to moderate increase in intra-epithelial hyaline droplets in all the male groups given Homosalate and in a few of the affected animals an increase in foci of basophilic (regenerating) tubules, single cell death and/or the granular casts. In liver, minimal or mild centrilobular hypertrophy of hepatocytes, consistent in appearance with enzyme induction, a male given 120 mg/kg bw/day and both sexes given 300 mg/kg bw/day or 750 mg/kg bw/day. In thymus, a greater incidence and severity of decreased cortical lymphocytes in males given 300 mg/kg bw/day and both sexes given 750 mg/kg bw/day. In thyroid glands, a greater incidence and/or severity of diffuse hypertrophy of the follicular epithelium in females given 300 mg/kg bw/day and in both sexes given 750 mg/kg bw/day. Reproduction and Breeding Data At the dose level of 750 mg/kg bw/day in males, significant changes in sperm morphology and reduction in sperm motility were noted. These effects were considered to be test item-related and adverse. At the dose level of 750 mg/kg bw/day, only one female had living pup at first litter check. A lower number of corpora luteaand increased post implantation loss were noted in this female if compared to values in the control group. No birth was recorded for the two remaining pregnant females at this dose level. At termination, only implantation sites were found in one of them and fetuses in uterus in the other. Consequently, 100% of pregnant females at the high dose level was affected with mortality of fetuses and / or newborns. Although due to a low number of pregnant females this conclusion was not supported by statistically meaningful evidence, based on these observations, a test item-related effect on fertility and gestation at the high-dose level should be considered. At the dose level of 300 mg/kg bw/day, higher incidence of post-implantation loss was noted. This effect was considered to be test item-related. Despite the increased post-implantation loss, litter size at this the dose level o was not reduced due to a higher number of implantation sites at this dose level when compared to the control group. At the dose levels of 60 and 120 mg/kg bw/day no indication of a test item-related effect on reproduction was noted. Litter size was considered not to be affected by the treatment with the test item up to the dose level of 300 mg/kg bw/day. No test item-related findings were noted in pups during first litter check and during lactation at any dose level. At the dose level of 750 mg/kg bw/day, on day 1 of the lactation period body weigh of one pup in litter no. 95 was lower compared to mean pup body weights in the control group. No effects on pup body weights were noted up to the dose level of 300 mg/kg bw/day. Summary: Based on adverse effects on food consumption and body weights in both sexes and mortality of females noted at the dose level of 750 mg/kg bw/day, a NOAEL (No Observed Adverse Effect Level) for general toxicity was established at 300 mg/kg bw/day for both sexes. At the dose level of 750 mg/kg bw/day test item-related effects on male fertility were noted: changes in sperm morphology and sperm motility correlating with reduced weights of prostate and seminal vesicles. Reproduction was affected down to the dose level of 300 mg/kg bw/day, where increased post implantation loss was noted. Under the conditions of this study, no indication of any effect on reproduction was noted at the dose levels of 60 and 120 mg/kg bw/day. However, because of low numbers of pregnant females, none of these dose levels could be conclusively confirmed as NOAEL.