Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2008 to 07 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study, no restrictions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han:HsdRccHan:WIST
- Source: Harlan UK Ltd, Oxon, UK
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: Males: 269 - 327 g; Females: 183 - 224 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and soft wood flake bedding (Harlan UK Ltd, Oxon, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (ad libitum): Pelleted Rodent 2018C Teklad Global (Certified) Diet, Harlan UK, Ltd. Oxon, UK)
- Water (ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment: Wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- The temperature and relative humidity controls were set to achieve target values.
- Temperature (°C): 21 ± 2
- Humidity (%): 55± 15
- Air changes (per hr): At least 15
- Photoperiod: 12 hrs dark /12 hrs light
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Route = Oral

PREPARATION OF DOSING SOLUTIONS
- Test material was prepared at appropriate concentrations as a suspension in the vehicle, Arachis oil BP.
- Concentrations made were 0, 2.5, 12.5 and 62.5 mg/mL
- No further details on the vehicle reported.

DOSE VOLUME
- 4 mL/kg bw/day
- The volume administered to each animal was based on the most recent body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating referred to as day 0 of pregnancy.
- Further matings: Not required


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test material formulation and analysed for concentration of zinc 3,5-bis(α-methylbenzyl)salicylate at Safepharm Analytical Laboratory using high performance liquid chromatography (HPLC) and external standard technique. The homogeneity and stability of the 2.5 mg/mL and 62.5 mg/mL formulations was confirmed
for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark. The achieved concentrations of the weekly formulations at all concentrations were considered acceptable.
Duration of treatment / exposure:
Males until day 43, females until day 5 post partum (up to 54 days)
Frequency of treatment:
Once daily
Details on study schedule:
Allocation of Animals to the Study:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
v) The males were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.
Remarks:
Doses / Concentrations:
0, 10, 50 and 250 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected on the basis of a preliminary seven day repeated dose range finding study which investigated dose levels of 250, 500 and 1000 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day. Treatment-related deaths occurred at 500 and 1000 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and 1-5 hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- On Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Body weights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
- Weekly consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
- Weekly food efficiency (body weight gain/food intake) was calculated for males and females during the pre-mating phase and during the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation or during lactation.

WATER CONSUMPTION: Yes
- Measured daily by weighing of water bottles.

REPRODUCTIVE PERFORMANCE: Yes.
The following were recorded:
- Date of mating
- Pre-coital interval
- Date and time of observed start of parturition
- Date and time of observed completion of parturition
- Duration of gestation



Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo.

For each litter the following was recorded:
i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Clinical condition of offspring from birth to Day 5 post partum
iv) Individual offspring and litter weights on Day 1 and 4 post partum

All live offspring were assessed for surface righting reflex on day 1 post partum.
Postmortem examinations (parental animals):
TERMINATION and NECROPSY
- Adult animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination, males on Day 43 and females on Day 5 post partum. Any female that failed to achieve pregnancy or to produce a litter was killed on or after Day 25 post coitum.
- All animals were given a full external and internal examination and any macroscopic abnormalities were recorded.
- For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy.

ORGAN WEIGHTS
- Males: Epididymides and testes

HISTOPATHOLOGY
- Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin (*Preserved in Bouin’s fluid and then in 70% IMS approximately 48 hours): Coagulating gland, seminal vesicles, epididymides *, testes*, prostate, ovaries, uterus/cervix/vagina, pituitary, gross lesions.
- All tissues were despatched to RCC Ltd, Zelgliweg 1, CH-4452 Itingen, Switzerland (Principal Investigator: K Weber). The tissues from control and 250 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.

Postmortem examinations (offspring):
TERMINATION and NECROPSY
- Surviving offspring were terminated via intra cardiac overdose of sodium pentobarbitone.
- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
The following parameters were subjected to statistical analysis:
- Body weight and body weight change
- Food consumption for females during gestation and lactation
- Litter data
- Implantation losses and viability indices
- Offspring body weight and body weight change
- Offspring surface righting
- Adult absolute and body weight relative organ weights

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pair wise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non - parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (p) are presented as follows:
p< 0.001***
p< 0.01**
p< 0.05*
p< 0.1 (*)
p ≥ 0.05 (not significant)
Reproductive indices:
- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Mating Index (%) = Number of animals mated x 100 / Number of animals paired
- Pregnancy Index (%) = Number of pregnant females x 100 / Number of animals mated
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index (%) = Number of females delivering live offspring x 100 / Number of pregnant females
Offspring viability indices:
- % pre- implantation loss = Number of corpora lutea - Number of implantation sites x 100 / Number of corpora lutea
- % post- implantation loss = Number of implantation sites - Total number of offspring born x 100 / Number of implantation sites
- Live Birth Index (%) = Number of offspring alive on day 1 x 100 / Number of offspring born  
- Viability Index (%) =Number of offspring alive on day 4 x 100 / Number of offspring alive on day 1
- Sex Ratio (% males) for days 1 & 4 post partum: Number of male offspring x 100 / Total number of offspring
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
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
MORTALITY AND CLINICAL SIGNS
Mortality: One female treated with 250mg/kg/day was killed in extremis on Day 2 post partum following severe clinical signs (hunched posture, lethargy, decreased respiration and ptosis). There were no further unscheduled deaths.
Clinical signs: Incidents of increased salivation were detected soon after dosing from Day 3 for animals of either sex treated with 250mg/kg/day, with transient incidents of increased salivation also detected up to one hour after dosing and this was also observed up to five hours after dosing on one occasion. Isolated instances of noisy respiration, gasping respiration, wet fur and associated staining around the mouth were detected for males and isolated instances of noisy respiration were also evident for two females treated at this dose level.
Similar clinical signs were evident at 50mg/kg/day albeit at a lesser incidence than that observed at 250mg/kg/day. Incidents of increased salivation were detected soon after dosing, and on occasion, up to one hour after dosing for animals of either sex treated at this dose level.
At 10mg/kg/day, one female displayed increased salivation soon after dosing on Day 37 and isolated instances of increased salivation following dosing were detected for some males during the treatment period.

BODY WEIGHT
Males treated with 250mg/kg/day displayed a reduction in body weight gain during the first week of treatment (p<0.001) when compared to controls, and a slight reduction in body weight gain was also evident during Week 3 although statistical significance was not achieved. These reductions resulted in lower cumulative body weight gains throughout the study (p<0.01- p<0.001) and lower mean body weights during the final two weeks of treatment when compared to controls (p<0.05). No adverse effects on body weight change were evident for males treated with 50 or 10 mg/kg/day.
Actual body weight losses were evident for a majority of the females treated with 250mg/kg/day during Week 1, resulting in a statistically significant reduction in body weight gains when compared to controls (p<0.001), resulting in a reduction in cumulative body weights gains for 250 mg/kg/day when compared to controls during Week 1 (p<0.001) and Week 2 (p<0.01).
A reduction in cumulative body weight gains was also evident for females treated with 50 mg/kg/day when compared to controls at the end of the maturation phase, however, the statistical significance was minimal (Week 2: p<0.05), therefore, this reduction was considered not to represent an adverse effect of treatment. Slight reduction in body weight change was also evident for females treated with 50mg/kg/day during the first two weeks of the study.
No adverse effects on body weight change were evident for females treated with10mg/kg/day during maturation.
No adverse effects on body weight change were evident for treated females throughout the gestation or lactation phases of the study, when compared to controls.

FOOD CONSUMPTION
Males treated with 250mg/kg/day displayed slight reductions in dietary intake, in comparison to controls, during the first two weeks of treatment with statistical significance achieved during Week 2 (p<0.05). A very slight reduction in dietary intake was also noticed during week 5. Food efficiencies (the ratio of body weight change to dietary intake) were lower than controls during Week 1 and Week 5. No adverse effects on dietary intake or food efficiency were evident for males treated with 50 or 10mg/kg/day.
During the first week of treatment, females treated with 250mg/kg/day showed a slightly lower dietary intake when compared to controls. Food efficiencies were similarly reduced during Week 1. No such effects were evident thereafter and dietary intake was comparable to controls during the gestation and lactation phases. No adverse effect of food efficiency was evident during the remainder of the maturation phase, or during early gestation.
No adverse effects on dietary intake or food efficiency were evident for females treated with 50 and 10mg/kg/day.

WATER CONSUMPTION
A substantial increase in water intake was evident for males treated with 250mg/kg/day during the pre-mating phase. During the first week of treatment, a statistically significant increase in mean weekly intake was evident when compared to controls (p<0.001). Statistical analysis of the mean daily values showed statistically significant increases on Day 3 (p<0.01) and Day 7 (p<0.05). Mean weekly water intake during the second week of treatment was still substantially higher for males treated with 250mg/kg/day when compared to controls (p<0.001). There were no water consumption measurements performed during the mating phase, however, a statistically significant increase in water intake was evident for males from all treated groups when compared to controls during Week 5 (p<0.001) and males treated with 250mg/kg/day also showed an increase in water intake during the final week of treatment (p<0.001).
A statistically significant increase in water intake was evident for females during the first week of treatment when compared to control (p<0.001) and values were still higher than controls during the second week of treatment (p<0.05). No adverse effects on water intake were detected during the gestation or lactation phases for 250 mg/kg/day females, or throughout the treatment period for females treated with 50 or 10 mg/kg/day.

REPRODUCTIVE PERFORMANCE
- No adverse effect on mating performance was detected. With the exception of one pair of animals treated with 50mg/kg/day, which mated after 12 days of pairing, all remaining animals from the control and treated groups mated within four days of pairing.
- Of the ten females from the 250mg/kg/day, only five females eventually produced live litters. No adverse effect on fertility was evident in the remaining dose groups and all females went on to give birth resulting in live litters.
- There was no adverse effect on gestation lengths between treated and control females. Females from all treatment groups went on to give birth following 22.5 to 23.5 days of gestation.
- All females from the control, 10 or 50mg/kg/day dose groups delivered offspring. In the 250 mg/kg/day dose group, although all animals mated, only five females produced litters. Three females were non-pregnant; two females displayed corpora lutea and implantation sites but did not produce offspring. Of the five pregnancies at 250mg/kg/day, one litter displayed a total litter loss on Day 2 post partum. Another litter showed dead offspring and the remaining offspring and dam were of poor physical health on Day 2 post partum, resulting in the termination of the dam and remaining litter. The remaining three litters and dams were unaffected.

GROSS PATHOLOGY
A majority of males treated with 250 mg/kg/day (7/10) displayed small seminal vesicles and one male also displayed small testes. One male treated with 50mg/kg/day and two males treated with 10mg/kg/day also displayed small seminal vesicles. For females, the interim death female treated with 250 mg/kg/day displayed a thickened glandular region of the stomach and a raised limiting ridge. One female treated with 250 mg/kg/day displayed thickened glandular epithelia and one female displayed a raised limiting ridge at terminal kill. A thickened glandular region of the stomach was also detected for one female treated with 50 mg/kg/day and for three females treated with 10 mg/kg/day. There were no other macroscopic findings considered to be attributable to treatment.

Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced body weight and food intake, increased water intake at 250 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced number of females with live foetuses at 250 mg/kg bw/day
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY
- A statistically significant reduction in litter size was evident for litters from the 250mg/kg/day when compared to control litters (p<0.01).
- No differences in sex ratio were evident for litters from treated animals when compared to controls.
- There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls, and pre-implantation losses from treated animals were comparable to controls. The high standard deviation detected for pre-implantation losses in the controls was attributed to two animals showing complete unilateral implantation loss.
- Although statistical significance was not achieved, higher percentage post-implantation losses were evident for females from the 250 mg/kg/day dose groups (41.1%) compared to percentage post- implantation losses from the controls (7.9%).
- Offspring viability on Day 4 at 250 mg/kg/day was lower than controls (60% and 99.4% respectively), although statistical significance was not achieved. No adverse effects on litter size or viability were evident at 50 or 10mg/kg/day.

CLINICAL SIGNS
At 250mg/kg/day, one litter revealed one dead female, one offspring was missing and one male displayed a physical injury to the tail on post partum Day 1. Total litter loss was evident on post partum Day 2. Another litter from this dose group revealed five dead offspring on Day 2 post partum. The remaining four offspring from this litter were reported as cold, weak and there was no milk present in the stomachs. The dam was also observed to be in poor physical health. Due to these observations, the dam and remaining litter were terminated on Day 2 post partum .There were no clinical signs or deaths in the remaining three litters from the 250mg/kg/day dose group. No clinically observable signs of toxicity were evident at 50 or 10mg/kg/day.

BODY WEIGHT
Reduced litters weights were evident from the 250mg/kg/day dose group when compared to controls however, statistical significance was not achieved. Mean body weights for male and female offspring from the 250 mg/kg/day females were less than those from the controls although statistical significance was only achieved for females at Day1 post partum (p<0.05) and at Day 4 post partum (p<0.001). Reduced body weight gains were also evident for both males and females from the 250 mg/kg/day group, in comparison to offspring from the control group and actual body weight losses were also evident at Day 4 post partum, although statistical significance was not achieved. No adverse effects on litter weights or body weights were evident at 50 or 10mg/kg/day.

SURFACE RIGHTING
Offspring from the 250mg/kg/day dose group showed a statistically significant reduction in passing the surface righting assessment on Day 1 post partum, when compared to controls (p<0.01). No adverse effect on surface righting was evident at 50 and 10mg/kg/day in comparison to offspring from controls.
Dose descriptor:
NOAEL
Remarks:
offspring toxicity
Generation:
F1a
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased post-implantation loss, reduced litter size, pup viability and pup body weight at 250 mg/kg bw/day
Reproductive effects observed:
not specified

Table 1: Incidence of increased salivation immediately after dosing

Days

Dose level (mg/kg bw/day)

Males

Females

0

10

50

250

0

10

50

250

1-7

0/10

0/10

0/10

2/10

0/10

0/10

0/10

3/10

8-14

0/10

1/10

4/10

10/10

0/10

0/10

0/10

10/10

15-21

0/10

1/10

6/10

10/10

0/10

0/10

0/10

10/10

22-28

0/10

0/10

8/10

10/10

0/10

0/10

0/10

9/10

29-35

0/10

2/10

6/10

10/10

0/10

0/10

7/10

10/10

36-42

0/10

4/10

6/10

10/10

0/10

1/10

7/10

10/10

 

Table 2: Body weight and body weight gain (g) during the maturation period

Day

Dose level (mg/kg bw/day)

Males

Females

0

10

50

250

0

10

50

250

1

298

300

297

299

206

203

205

205

8

319

323

317

306

211

208

207

202

15

337

342

334

318

217

210

210

207

Gain 1-15

38

41

36

19***

12

7

5*

2**

* Statistically significant difference from control group (p> 0.05)

*** Statistically significant difference from control group (p> 0.001)

 

Table 3: Food consumption (g/rat/day) during the maturation period

Week

Dose level (mg/kg bw/day)

Males

Females

0

10

50

250

0

10

50

250

1

21

21

20

20

15

14

14

12

2

20

20

19

17*

13

13

13

13

* Statistically significant difference from control group (p> 0.05)

 

Table 4: Water consumption (g/rat/day) during the maturation period

Week

Dose level (mg/kg bw/day)

Males

Females

0

10

50

250

0

10

50

250

1

25.3

26.5

25.9

39.4***

19.7

20.4

19.0

26.1***

2

26.0

27.8

27.8

32.4***

20.1

18.9

19.1

23.5*

* Statistically significant difference from control group (p> 0.05)

*** Statistically significant difference from control group (p> 0.001)

 

Table 5: Reproductive performance – selected endpoints

Endpoint

Dose level (mg/kg bw/day)

0

10

50

250

Mating index (%)

100

100

100

100

Pregnancy index (%)

100

100

100

70

Parturition index (%)

100

100

100

71.4

Females with live offspring

10/10

10/10

10/10

5/10

 

Table 6: Implantation loss and offspring survival indices

Index

Dose level (mg/kg bw/day)

0

10

50

250

Pre-implantation loss (%)

23.8

19.9

10.3

33.8

Post-implantation loss (%)

7.9

4.5

7.1

41.1

Live birth index (%)

99.3

98.3

98.4

100

Viability index (%)

99.4

99.1

99.2

60.0

 

Table 7: Litter data

Endpoint

Dose level (mg/kg bw/day)

0

10

50

250

Number live day 1

10.7

10.7

12.2

8.0**

Number live day 4

10.6

10.6

12.1

9.0**

Body weight (g) day 1 - males

6.4

6.0

6.0

5.5

Body weight (g) day 1 - females

6.2

5.8

5.7

5.1*

Body weight (g) day 4 - males

9.7

8.7

8.7

8.5

Body weight (g) day 4 - females

9.5

8.5

8.3

8.9***

* Statistically significant difference from control group (p> 0.05)

** Statistically significant difference from control group (p> 0.01)

*** Statistically significant difference from control group (p> 0.001)

 

Conclusions:
The NOAEL for parental, offspring and reproductive toxicity was 50 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.
Executive summary:

Zinc 3,5-bis(α-methylbenzyl)salicylate was administered by gavage to 3 groups each of 10 male and 10 female WistarHan:HsdRccHan:WIST rats, for up to 54 consecutive days, (including a two week maturation phase, pairing phase, gestation and early lactation for females) at dose levels of 10, 50 and 250 mg/kg/day. A control group of 10 males and 10 females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight, food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a 1 male: 1 female basis within each treatment group, on Day 15 of the study. Females were allowed to litter and rear their offspring to Day 5 post partum. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed on the control and high dose group parental animals.

 

Increased salivation was detected soon after dosing for animals treated with 250 mg/kg/day, from Day 3 onwards, with the effect still evident up to one hour following dosing. Increased salivation was also detected for animals treated with 50 or 10 mg/kg/day, albeit to a lesser extent. Slight reductions in body weight gains were evident for females treated with 250 or 50 mg/kg/day during the first two weeks of treatment. Males treated with 250 mg/kg/day also displayed reductions in body weight gains throughout the treatment period when compared to controls. No adverse effects were detected for males treated with 50 or 10 mg/kg/day, for females treated with 50 or 10 mg/kg/day during the gestation and lactation phases of the study, or for females treated with 10 mg/kg/day throughout the treatment period.

 

Reduced dietary intake and food efficiency were evident for females treated with 250 mg/kg/day during the first week of treatment. Males treated with 250 mg/kg/day displayed a slight reduction in dietary intake during the first two weeks and during week 5 of the study. Food efficiencies were similarly reduced. No adverse effects were detected for animals treated with 50 or 10 mg/kg/day. An increase in water intake was evident for males treated with 250 mg/kg/day when compared to controls throughout the treatment period. Slight increases in water intake were evident for 250 mg/kg/day females when compared to controls during the maturation phase. No overt differences in water intake were evident during the gestation and lactation phases of the study. No adverse effects on water intake were detected for animals treated with 50 or 10 mg/kg/day.

 

There were no intergroup differences in mating performance or gestation lengths. Pregnancy was achieved for all control, 10 and 50 mg/kg/day females. However, only five out of ten females treated with 250 mg/kg/day produced litters. From evaluation of the corpora lutea and implantation data, the percentage post-implantation loss at 250 mg/kg/day was much higher when compared to the control value. Reduced litter sizes were evident for females treated with 250 mg/kg/day when compared to controls and also, the number of viable litters was lower. There was no obvious difference in the sex ratio of the offspring. No adverse effects on litter size and viability were evident from the 50 or 10 mg/kg/day dose groups. Mean male and female offspring body weights were lower from females treated with 250 mg/kg/day when compared to controls. No adverse effects on body weight were evident for litters from the 50 or 10 mg/kg/day dose groups.

 

The NOAEL for parental, offspring and reproductive toxicity was 50 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is categorised as Klimisch 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Zinc 3,5-bis(α-methylbenzyl)salicylate: Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat SPL Project Number: 1139/0106: The oral administration of zinc 3,5-bis(α-methylbenzyl)salicylate to rats for a period of up to fifty-four consecutive days at dose levels of up to 250mg/kg/day, resulted in treatment-related systemic and consequently reproductive effects at 250 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic and reproductive toxicity was therefore considered to be 50 mg/kg/day.

There were no intergroup differences in mating performance or gestation lengths. Pregnancy was achieved for all control, 10 and 50 mg/kg/day females. However, only five out of ten females treated with 250 mg/kg/day produced litters. From evaluation of the corpora lutea and implantation data, the percentage post-implantation loss at 250 mg/kg/day was much higher when compared to the control value. Reduced litter sizes were evident for females treated with 250 mg/kg/day when compared to controls and also, the number of viable litters was lower. There was no obvious difference in the sex ratio of the offspring. No adverse effects on litter size and viability were evident from the 50 or 10 mg/kg/day dose groups. Mean male and female offspring body weights were lower from females treated with 250 mg/kg/day when compared to controls. No adverse effects on body weight were evident for litters from the 50 or 10 mg/kg/day dose groups.


Short description of key information:
Zinc 3,5-bis(α-methylbenzyl)salicylate: Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat SPL Project Number: 1139/0106: The oral administration of zinc 3,5-bis(α-methylbenzyl)salicylate to rats for a period of up to fifty-four consecutive days at dose levels of up to 250mg/kg/day, resulted in treatment-related systemic and consequently reproductive effects at 250 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic and reproductive toxicity was therefore considered to be 50 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Only 1 study is available.

Effects on developmental toxicity

Description of key information
Zinc 3,5-bis(α-methylbenzyl)salicylate: Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat SPL Project Number: 1139/0106: The oral administration of zinc 3,5-bis(α-methylbenzyl)salicylate to rats for a period of up to fifty-four consecutive days at dose levels of up to 250mg/kg/day, resulted in treatment-related systemic and consequently reproductive effects at 250 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic and reproductive toxicity was therefore considered to be 50 mg/kg/day. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2008 to 07 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study, no restrictions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han:HsdRccHan:WIST
- Source: Harlan UK Ltd, Oxon, UK
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: Males: 269 - 327 g; Females: 183 - 224 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and soft wood flake bedding (Harlan UK Ltd, Oxon, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (ad libitum): Pelleted Rodent 2018C Teklad Global (Certified) Diet, Harlan UK, Ltd. Oxon, UK)
- Water (ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment: Wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- The temperature and relative humidity controls were set to achieve target values.
- Temperature (°C): 21 ± 2
- Humidity (%): 55± 15
- Air changes (per hr): At least 15
- Photoperiod: 12 hrs dark /12 hrs light
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Route = Oral

PREPARATION OF DOSING SOLUTIONS
- Test material was prepared at appropriate concentrations as a suspension in the vehicle, Arachis oil BP.
- Concentrations made were 0, 2.5, 12.5 and 62.5 mg/mL
- No further details on the vehicle reported.

DOSE VOLUME
- 4 mL/kg bw/day
- The volume administered to each animal was based on the most recent body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test material formulation and analysed for concentration of zinc 3,5-bis(α-methylbenzyl)salicylate at Safepharm Analytical Laboratory using high performance liquid chromatography (HPLC) and external standard technique. The homogeneity and stability of the 2.5 mg/mL and 62.5 mg/mL formulations was confirmed for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark. The achieved concentrations of the weekly formulations at all concentrations were considered acceptable.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating referred to as day 0 of pregnancy.
- Further matings: Not required
Duration of treatment / exposure:
Males until day 43, females until day 5 post partum (up to 54 days)
Frequency of treatment:
Once daily
Duration of test:
7 weeks
Remarks:
Doses / Concentrations:
0, 10, 50 and 250 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose level selection:
The dose levels were selected on the basis of a preliminary seven day repeated dose range finding study which investigated dose levels of 250, 500 and 1000 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day. Treatment-related deaths occurred at 500 and 1000 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.
Allocation of Animals to the Study:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
v) The males were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and 1-5 hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends.
BODY WEIGHT: Yes
- On Day 1 (prior to dosing) and then weekly for females until mating was evident. Body weights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
- Weekly consumption was recorded for each cage of adults until pairing. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
- Weekly food efficiency (body weight gain/food intake) was calculated during the pre-mating phase and during the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation or during lactation.

WATER CONSUMPTION: Yes
- Measured daily by weighing of water bottles.

REPRODUCTIVE PERFORMANCE: Yes.
The following were recorded:
- Date of mating
- Pre-coital interval
- Date and time of observed start of parturition
- Date and time of observed completion of parturition
- Duration of gestation

TERMINATION and NECROPSY
- Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any female that failed to achieve pregnancy or to produce a litter was killed on or after Day 25 post coitum.
- All animals were given a full external and internal examination and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY
- Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin: ovaries, uterus/cervix/vagina, pituitary. gross lesions.
- All tissues were despatched to RCC Ltd, Zelgliweg 1, CH-4452 Itingen, Switzerland (Principal Investigator: K Weber). The tissues from control and 250 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes (This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution.)
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
No examination of foetuses as females were allowed to litter.
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo.
For each litter the following was recorded:
i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Clinical condition of offspring from birth to Day 5 post partum
iv) Individual offspring and litter weights on Day 1 and 4 post partum

All live offspring were assessed for surface righting reflex on day 1 post partum.
Statistics:
The following parameters were subjected to statistical analysis:
- Body weight and body weight change
- Food consumption for females during gestation and lactation
- Litter data
- Implantation losses and viability indices
- Offspring body weight and body weight change
- Offspring surface righting
- Adult absolute and body weight relative organ weights

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pair wise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non - parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (p) are presented as follows:
p< 0.001***
p< 0.01**
p< 0.05*
p< 0.1 (*)
p ≥ 0.05 (not significant)
Indices:
- % pre- implantation loss = Number of corpora lutea - Number of implantation sites x 100 / Number of corpora lutea
- % post- implantation loss = Number of implantation sites - Total number of offspring born x 100 / Number of implantation sites
- Live Birth Index (%) = Number of offspring alive on day 1 x 100 / Number of offspring born
- Viability Index (%) =Number of offspring alive on day 4 x 100 / Number of offspring alive on day 1
- Sex Ratio (% males) for days 1 & 4 post partum: Number of male offspring x 100 / Total number of offspring
Historical control data:
No
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
Mortality: One female treated with 250mg/kg/day was killed in extremis on Day 2 post partum following severe clinical signs (hunched posture, lethargy, decreased respiration and ptosis). There were no further unscheduled deaths.
Clinical signs: Incidents of increased salivation were detected soon after dosing from Day 3 for females treated with 250mg/kg/day, with transient incidents of increased salivation also detected up to one hour after dosing and this was also observed up to five hours after dosing on one occasion. Isolated instances of noisy respiration were also evident for two females treated at this dose level.
Similar clinical signs were evident at 50mg/kg/day albeit at a lesser incidence than that observed at 250mg/kg/day. Incidents of increased salivation were detected soon after dosing, and on occasion, up to one hour after dosing.
At 10mg/kg/day, one female displayed increased salivation soon after dosing on Day 37.

BODY WEIGHT
Actual body weight losses were evident for a majority of the females treated with 250mg/kg/day during Week 1, resulting in a statistically significant reduction in body weight gains when compared to controls (p<0.001), resulting in a reduction in cumulative body weights gains for 250 mg/kg/day when compared to controls during Week 1 (p<0.001) and Week 2 (p<0.01).
A reduction in cumulative body weight gains was also evident for females treated with 50 mg/kg/day when compared to controls at the end of the maturation phase, however, the statistical significance was minimal (Week 2: p<0.05), therefore, this reduction was considered not to represent an adverse effect of treatment. Slight reduction in body weight change was also evident for females treated with 50mg/kg/day during the first two weeks of the study.
No adverse effects on body weight change were evident for females treated with10mg/kg/day during maturation.
No adverse effects on body weight change were evident for treated females throughout the gestation or lactation phases of the study, when compared to controls.

FOOD CONSUMPTION
During the first week of treatment, females treated with 250mg/kg/day showed a slightly lower dietary intake when compared to controls. Food efficiencies were similarly reduced during Week 1. No such effects were evident thereafter and dietary intake was comparable to controls during the gestation and lactation phases. No adverse effect of food efficiency was evident during the remainder of the maturation phase, or during early gestation.
No adverse effects on dietary intake or food efficiency were evident for females treated with 50 and 10mg/kg/day.

WATER CONSUMPTION
A statistically significant increase in water intake was evident for females during the first week of treatment when compared to control (p<0.001) and values were still higher than controls during the second week of treatment (p<0.05). No adverse effects on water intake were detected during the gestation or lactation phases for 250 mg/kg/day females, or throughout the treatment period for females treated with 50 or 10 mg/kg/day.

REPRODUCTIVE PERFORMANCE
- No adverse effect on mating performance was detected. With the exception of one pair of animals treated with 50mg/kg/day, which mated after 12 days of pairing, all remaining animals from the control and treated groups mated within four days of pairing.
- Of the ten females from the 250mg/kg/day, only five females eventually produced live litters. No adverse effect on fertility was evident in the remaining dose groups and all females went on to give birth resulting in live litters.
- There was no adverse effect on gestation lengths between treated and control females. Females from all treatment groups went on to give birth following 22.5 to 23.5 days of gestation.
- All females from the control, 10 or 50mg/kg/day dose groups delivered offspring. In the 250 mg/kg/day dose group, although all animals mated, only five females produced litters. Three females were non-pregnant; two females displayed corpora lutea and implantation sites but did not produce offspring. Of the five pregnancies at 250mg/kg/day, one litter displayed a total litter loss on Day 2 post partum. Another litter showed dead offspring and the remaining offspring and dam were of poor physical health on Day 2 post partum, resulting in the termination of the dam and remaining litter. The remaining three litters and dams were unaffected.

GROSS PATHOLOGY
The interim death female treated with 250 mg/kg/day displayed a thickened glandular region of the stomach and a raised limiting ridge. One female treated with 250 mg/kg/day displayed thickened glandular epithelia and one female displayed a raised limiting ridge at terminal kill. A thickened glandular region of the stomach was also detected for one female treated with 50 mg/kg/day and for three females treated with 10 mg/kg/day. There were no other macroscopic findings considered to be attributable to treatment.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY
- A statistically significant reduction in litter size was evident for litters from the 250mg/kg/day when compared to control litters (p<0.01).
- No differences in sex ratio were evident for litters from treated animals when compared to controls.
- There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls, and pre-implantation losses from treated animals were comparable to controls. The high standard deviation detected for pre-implantation losses in the controls was attributed to two animals showing complete unilateral implantation loss.
- Although statistical significance was not achieved, higher percentage post-implantation losses were evident for females from the 250 mg/kg/day dose groups (41.1%) compared to percentage post- implantation losses from the controls (7.9%).
- Offspring viability on Day 4 at 250 mg/kg/day was lower than controls (60% and 99.4% respectively), although statistical significance was not achieved. No adverse effects on litter size or viability were evident at 50 or 10mg/kg/day.

CLINICAL SIGNS
At 250mg/kg/day, one litter revealed one dead female, one offspring was missing and one male displayed a physical injury to the tail on post partum Day 1. Total litter loss was evident on post partum Day 2. Another litter from this dose group revealed five dead offspring on Day 2 post partum. The remaining four offspring from this litter were reported as cold, weak and there was no milk present in the stomachs. The dam was also observed to be in poor physical health. Due to these observations, the dam and remaining litter were terminated on Day 2 post partum .There were no clinical signs or deaths in the remaining three litters from the 250mg/kg/day dose group. No clinically observable signs of toxicity were evident at 50 or 10mg/kg/day.

BODY WEIGHT
Reduced litters weights were evident from the 250mg/kg/day dose group when compared to controls however, statistical significance was not achieved. Mean body weights for male and female offspring from the 250 mg/kg/day females were less than those from the controls although statistical significance was only achieved for females at Day1 post partum (p<0.05) and at Day 4 post partum (p<0.001). Reduced body weight gains were also evident for both males and females from the 250 mg/kg/day group, in comparison to offspring from the control group and actual body weight losses were also evident at Day 4 post partum, although statistical significance was not achieved. No adverse effects on litter weights or body weights were evident at 50 or 10mg/kg/day.

SURFACE RIGHTING
Offspring from the 250mg/kg/day dose group showed a statistically significant reduction in passing the surface righting assessment on Day 1 post partum, when compared to controls (p<0.01). No adverse effect on surface righting was evident at 50 and 10mg/kg/day in comparison to offspring from controls.
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Incidence of increased salivation immediately after dosing

Days

Dose level (mg/kg bw/day)

0

10

50

250

1-7

0/10

0/10

0/10

3/10

8-14

0/10

0/10

0/10

10/10

15-21

0/10

0/10

0/10

10/10

22-28

0/10

0/10

0/10

9/10

29-35

0/10

0/10

7/10

10/10

36-42

0/10

1/10

7/10

10/10

 

Table 2: Body weight and body weight gain (g) during the maturation period

Day

Dose level (mg/kg bw/day)

0

10

50

250

1

206

203

205

205

8

211

208

207

202

15

217

210

210

207

Gain 1-15

12

7

5*

2**

* Statistically significant difference from control group (p> 0.05)

** Statistically significant difference from control group (p> 0.01)

 

Table 3: Food consumption (g/rat/day) during the maturation period

Week

Dose level (mg/kg bw/day)

0

10

50

250

1

15

14

14

12

2

13

13

13

13

 

Table 4: Water consumption (g/rat/day) during the maturation period

Week

Dose level (mg/kg bw/day)

0

10

50

250

1

19.7

20.4

19.0

26.1***

2

20.1

18.9

19.1

23.5*

* Statistically significant difference from control group (p> 0.05)

*** Statistically significant difference from control group (p> 0.001)

 

Table 5: Implantation loss and offspring survival

Index

Dose level (mg/kg bw/day)

0

10

50

250

Females with live offspring

10/10

10/10

10/10

5/10

Pre-implantation loss (%)

23.8

19.9

10.3

33.8

Post-implantation loss (%)

7.9

4.5

7.1

41.1

Live birth index (%)

99.3

98.3

98.4

100

Viability index (%)

99.4

99.1

99.2

60.0

 

Table 6: Litter data

Endpoint

Dose level (mg/kg bw/day)

0

10

50

250

Number live day 1

10.7

10.7

12.2

8.0**

Number live day 4

10.6

10.6

12.1

9.0**

Body weight (g) day 1 - males

6.4

6.0

6.0

5.5

Body weight (g) day 1 - females

6.2

5.8

5.7

5.1*

Body weight (g) day 4 - males

9.7

8.7

8.7

8.5

Body weight (g) day 4 - females

9.5

8.5

8.3

8.9***

* Statistically significant difference from control group (p> 0.05)

** Statistically significant difference from control group (p> 0.01)

*** Statistically significant difference from control group (p> 0.001)

 

 

Conclusions:
The NOAEL for maternal and developmental toxicity was 50 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.
Executive summary:

Zinc 3,5-bis(α-methylbenzyl)salicylate was administered by gavage to 3 groups each of 10 male and 10 female WistarHan:HsdRccHan:WIST rats, for up to 54 consecutive days, (including a two week maturation phase, pairing phase, gestation and early lactation for females) at dose levels of 10, 50 and 250 mg/kg/day. A control group of 10 males and 10 females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight, food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a 1 male: 1 female basis within each treatment group, on Day 15 of the study. Females were allowed to litter and rear their offspring to Day 5 post partum. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed on the control and high dose group parental animals.

 

Increased salivation was detected soon after dosing for animals treated with 250 mg/kg/day, from Day 3 onwards, with the effect still evident up to one hour following dosing. Increased salivation was also detected for animals treated with 50 or 10 mg/kg/day, albeit to a lesser extent. Slight reductions in body weight gains were evident for females treated with 250 or 50 mg/kg/day during the first two weeks of treatment. No adverse effects were detected for females treated with 50 or 10 mg/kg/day during the gestation and lactation phases of the study, or for females treated with 10 mg/kg/day throughout the treatment period.

 

Reduced dietary intake and food efficiency were evident for females treated with 250 mg/kg/day during the first week of treatment. Food efficiencies were similarly reduced. No adverse effects were detected for animals treated with 50 or 10 mg/kg/day. Slight increases in water intake were evident for 250 mg/kg/day females when compared to controls during the maturation phase. No overt differences in water intake were evident during the gestation and lactation phases of the study. No adverse effects on water intake were detected for animals treated with 50 or 10 mg/kg/day.

 

There were no intergroup differences in mating performance or gestation lengths. Pregnancy was achieved for all control, 10 and 50 mg/kg/day females. However, only five out of ten females treated with 250 mg/kg/day produced litters. From evaluation of the corpora lutea and implantation data, the percentage post-implantation loss at 250 mg/kg/day was much higher when compared to the control value. Reduced litter sizes were evident for females treated with 250 mg/kg/day when compared to controls and also, the number of viable litters was lower. There was no obvious difference in the sex ratio of the offspring. No adverse effects on litter size and viability were evident from the 50 or 10 mg/kg/day dose groups. Mean male and female offspring body weights were lower from females treated with 250 mg/kg/day when compared to controls. No adverse effects on body weight were evident for litters from the 50 or 10 mg/kg/day dose groups.

 

The NOAEL for maternal and developmental toxicity was 50 mg zinc 3,5-bis(α-methylbenzyl)salicylate/kg bw/day.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is categorised as Klimisch 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

See Discussion under 'Effects on fertility' above.


Justification for selection of Effect on developmental toxicity: via oral route:
Only 1 study is available.

Justification for classification or non-classification

The above study has been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because it is a GLP study conducted according to a recognised test method. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

Recommendation on classification according to Regulation (EC) No 1272/2008

Zinc 3,5-bis(α-methylbenzyl)salicylate is associated with effects on female reproductive function when given orally to rats. However it is likely that these effects are a consequence of maternal toxicity rather than a direct perturbation of the reproductive system.

There is a clear no effect level (NOEL) for systemic toxicity at 50 mg/kg/day in the reproductive and developmental screen, while maternal effects were confined to the high dose group (250 mg/kg/day) in which clear evidence of systemic toxicity was seen.

In the males in the reproduction/developmental toxicity screening test there was some evidence of reduced size of the seminal vesicles and, in one male, small testes. However, these macroscopic observations were not reflected in group mean organ weights of the testes or epididymides (seminal vesicles were not weighed). Small seminal vesicles were also evident in two males each at 50 and 10 mg/kg/day. The reduction in seminal vesicle size at the two lower doses was not associated with any effect on fertility as expressed by maternal litter data. There were no histological findings in the testes or epididymides. In the seminal vesicles, reduced secretory content was seen in three males at the high dose but the report pointed out that this is very variable in laboratory rats and there was no convincing evidence that this was related to treatment.

Overall, it is considered that the reproductive effects in this study were attributable to systemic toxicity as they were seen only at the highest dose. The report made the point that the cause of the reduction in pregnancy rate at the highest dose could not be accurately elucidated from this study.

In the 90-day repeat-dose study a NOEL of 10 mg/kg/day was established; there was no evidence of histopathological effect related to the test material in any group. This study was conducted with a mixture containing 80% zinc 3,5-bis(α-methylbenzyl)salicylate, 20% methylstyrene and styrene polymer in rats, it is considered that there was sufficient zinc 3,5-bis(α-methylbenzyl)salicylate in the test-item to give an accurate representation of potential toxicity. In selecting doses for the reproduction/developmental toxicity screening test in the rat, the low dose was placed at 10 mg/kg/day, which was slightly higher than the NOEL noted in the 90-day study with the mixture. The high dose of 250 mg/kg/day was notably higher than the high dose in the 90-day study at which toxicity was seen and would reasonably be expected to be associated with more extensive toxicity than seen in the 90-day study.

This supports the contention that the high dose in the reproduction/developmental toxicity screening test would be associated with toxicity and that any effects on reproductive function would reasonably be attributed to this rather than to direct effect on reproduction.

Points to consider:

• Reproductive effects were only observed at maternally toxic doses in the reproductive/developmental toxicity screening study.

• No developmental abnormalities were detected in the reproductive/developmental toxicity screening study despite maternal toxicity.

• In both the reproductive/developmental toxicity screening study and the 90-day repeat dose study a decrease in body-weight was observed in the top dose (100 and 250 mg/kg/day respectively) potentially indicating systemic toxicity.

• No evidence was found in the pathology (either at necropsy or histopathology) of the reproductive/developmental toxicity screening or the 90-day repeat dose studies of treatment-related effects on the reproductive organs.

It is recommended that this substance should not be classified as a reproductive and developmental toxin as there is evidence that the reproductive effect observed are secondary to systemic toxicity.