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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 06 to November 05, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl salicylate
EC Number:
204-263-4
EC Name:
2-ethylhexyl salicylate
Cas Number:
118-60-5
Molecular formula:
C15H22O3
IUPAC Name:
2-ethylhexyl 2-hydroxybenzoate
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Details on test animals or test system and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
Number of Animals: 44 males: 11 per group 44 females: 11 per group

Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment): Males: 312 to 351 g Females: 208 to 244 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.
Husbandry
Room Numbers, Füllinsdorf: E0441A (acclimatization) and E0441 (after acclimatization) Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of ran-domization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg/Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/ lot nos. 100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

Diet: Pelleted standard Harlan Teklad 2018C (batch no. 43/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. .
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Four groups of 11 males and 11 females were treated by gavage with Neo Heliopan OS once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

Vehicle and Control Item
Identification: Corn oil
Source: Carl Roth GmbH
Batch Number: 292189296
Expiry Date (Retest Date): 02-Aug-2017
Storage Conditions: Room temperature (20 ± 5 °C)
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.

In conclusion, the results indicate the accurate use of the test item and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
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.
Duration of treatment / exposure:
Males: 28 days Females: Approximately 7 weeks
Frequency of treatment:
Once daily
Duration of test:
Approximately 7 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 80, 250 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group)
Group 2: 25 mg/kg/day
Group 3: 80 mg/kg/day
Group 4: 250 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D54872, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in mortality and adverse toxic effects at the dose level
of 1000 mg/kg bw/day and adverse toxic effects at the dose level of 300 mg/kg bw/day.
Dose Volume: 4 mL/kg body weight
Dose Concentrations: Group 1: 0.00 mg/mL
Group 2: 6.25 mg/mL
Group 3: 20.00 mg/mL
Group 4: 62.50 mg/mL
Duration of Acclimatization Period: Minimum 5 days
Duration of Treatment Period: Males: 28 days Females: Approximately 7 weeks

Examinations

Maternal examinations:
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 - 14; after pairing period weekly.
Females: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; 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.
Ovaries and uterine content:
Ovaries were checked for discoloration and uteri for fetus content and discoloration.
Fetal examinations:
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 possible), 1 and 4 post partum. Pups and dams were sacrificed on day 4 post partum. All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
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 [see References (2)] (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 [see References (3)] (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 [see References (4)] was applied if the variables could be dichotomized without loss of information.
Indices:
Reproductive indices recorded were fertility indices, mean precoital time, post-implantation loss, and offspring viability indices were mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 250 mg/kg bw/d slight but non-significant changes on weight gain were noted.

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
25 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

Details on embryotoxic / teratogenic effects:
Treatment with the test item at the dose levels of 250 and 80 mg/kg bw/day caused a reduction in gestation index (number of females with living pups as a percentage of females pregnant) as well as an increase in incidence of post-implantation loss resulting in a lower litter size. Further, at the dose levels of 250 and 80 mg/kg bw/day, prolonged gestation period was noted. These findings were considered to be test item-related adverse effects.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

1 Viability / Mortality

At the dose level of 250 mg/kg bw/day, one female (no. 78) was found dead on day 23 of the gestation period. During necropsy, fetuses were found in uterus of this female. A slight body weight loss was noted in this female on day 22 of the gestation period but no other signs and no macroscopical or microscopical findings indicating bad condition of this female were noted. Death of the female was most probably a result of difficult parturition and considered to be test-item related. All remaining animals of P generation survived the scheduled study period.

2 Clinical Signs or Observations

No test item-related findings were noted in males or females at any dose level. In one female (67) at the mid-dose level, hunched posture and ruffled fur were noted from day 2 of the lactation period onwards. Further, slight swelling in axillary region was observed in one female (no. 79) at the high-dose level from day 9 of the gestation period onwards. Because of isolated occurrence, these findings were considered not to be related to the treatment with the test item. No further findings were noted in males or females at any dose level.

3 Food Consumption of Males

The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/kg bw/day were respectively: +0.9%, -0.9% and +1.7% during the pre-pairing period (percentages refer to the respective values in the control group).

4 Food Consumption of Females Pre-Pairing, Gestation and Lactation Periods

At the dose levels of 250 and 80 mg/kg bw/day, reduced food consumption was noted during lactation. Mean food consumption was 16.4 and 19.8 g/animal/day at the dose levels of 250 and 80 mg/kg bw/day, respectively and 22.2 g/animal/day in the control group. Although the differences were not statistically significant, they were dose dependent and therefore considered to probably be related to the treatment with the test item. No effects on food consumption were noted at the dose level of 80 mg/kg bw/day. The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/ kg bw/day were respectively: -5.6%, -0.6% and -4.4% during the pre-pairing period, -2.4%, +6.2% and +4.8% during the gestation period and +1.4%, -10.8% and -26.1% during the lactation period (percentages refer to the respective values in the control group).

5 Body Weights of Males Pre-Pairing and Pairing Periods

At the dose level of 250 mg/kg bw/day, slight but statistically significant reduction in body weight gain was noted on day 13 of the pre-pairing period. Body weight gain was also slightly lower on further days at the end of this period but without statistical significance. No significant differences in absolute body weights were noted at this dose level. At the dose levels of 25 and 80 mg/kg bw/day, no test item-related effects on absolute body weights or body weight gain were noted. The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +15%, +15%, +13% and +13% during the pre-pairing period and +11%, +9%, +9% and +10% during the pairing period (percentages refer to the body weight gain within the period). At the low- and mid-dose levels, statistically significantly lower body weight gains were noted on individual days during the pairing period. Because the differences were minor and did not follow dose dependency, they were considered not to be related to the treatment with the test item.

6 Body Weights of Females Pre-Pairing, Pairing, Gestation and Lactation Periods

At the dose level of 250 mg/kg bw/day, statistically significant reduction in body weight gain was noted on day 4 of the lactation period. No significant differences in absolute body weights were noted at this dose level at any time. At the dose levels of 25 and 80 mg/kg bw/day, no effects on absolute body weights or body weight gain were noted. The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +8%, +7%, +9% and +6% during the pre-pairing period, +51%, +55%, +56% and +48% during the gestation period and +5%, +2%, +2% and ±0% during the lactation period (percentages refer to the body weight gain within the period).

Mating Performance and Fertility:

No effects on mating performance, fertility index or conception rate were noted at any dose level. No effects on gestation index were noted at the dose level of 25 mg/kg bw/day; it was 100% at this dose level.

Duration of Gestation :

At the dose levels of 250 and 80 mg/kg bw/day, prolonged gestation period was noted. Mean duration of gestation was 22.6 and 22.0 days at the dose levels of 250 and 80 mg/kg bw/day, respectively, compared to 21.5 days in the control group. Mean prolongation of the gestation at the high- and mid-dose levels was not statistically significant. However, it was dose dependent and the values were beyond the biological background (historical control data included values of gestation length from 21.2 to 21.8 days). Therefore this finding was considered to be test item-related. At the dose level of 25 mg/kg bw/day, mean gestation of duration was the same like the control value; 21.5 days and therefore not affected by the treatment. Corpora Lutea Count : No effects on corpora lutea count were observed at any dose level. Implantation Rate and Post-Implantation Loss : Number of implantations was not affected by the treatment with the test item at any dose level. At the dose level of 25 mg/kg bw/day, no effects on post-implantation loss were noted.

Applicant's summary and conclusion

Conclusions:
Based on the observation of increased post-implantation loss, reduction in gestation index and lower litter size, the NOAEL for developmental toxicity is detremined to be 25 mg/kg bw/d.
Executive summary:

This study was performed to generate preliminary information concerning the effects of test article on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. The test item was administered in corn oil as vehicle at dosages of 25, 80, and 250 mg/kg body weight/day, and controls received the vehicle only. Test article was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

At the high dose level, one female was found dead on day 23 of the gestation period which was considered to be a result of birth complications. No further adverse effects were noted in males or females at any dose level. At the dose levels of 250 and 80

mg/kg bw/day a reduction in gestation index as well as an increase in incidence of post-implantation loss resulting in a lower litter size were noted. These effects were statistically significant and dose dependent and therefore considered to be test itemrelated

and adverse. Based on the individual data, increased post-implantation loss occurred predominantly in females with prolonged gestation. A prolonged gestation was noted at the high- and mid-dose level, which were considered to be test itemrelated.

No compensation for lower body weights occurred during lactation. Reduction of pup absolute body weights was considered to be adverse. No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by the exposure to the test item at any dose level. Treatment with the test item at the dose level of 250

mg/kg bw/day caused a reduction in body weights of pups recorded on day 1 and 4 of the lactation period. During this period, body weight gain of pups at the high-dose level was similar to body weight gain of pups in the control group. Reduction in absolute body weights of pups was considered to be test item-related adverse effect. No test item-related effects on body weights or body weight gain of pups were noted at the dose levels of 25 and 80 mg/kg bw/day. No test item-related macroscopical findings were found in pups at any dose level.

Based on the observation of increased post-implantation loss, reduction in gestation index and lower litter size, the NOAEL for developmental toxicity is determined to be 25 mg/kg bw/d.