Reaction mass of α,α,4-trimethyl-, (1S)-, 3-cyclohexene-1-methanol and α,α,4-trimethyl-, (1R)-, 3-cyclohexene-1-methanol and 1-methyl-4-(1-methylethylidene)-cyclohexanol

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 11 to October 05, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Investigatory study succeeding OECD 422 screening test, performed in GLP laboratory.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Principles of method if other than guideline:

Investigatory study, succeeding OECD 422 screening test, was performed to compare the toxicity of Terpineol multiconstituent to the male reproductive system when administered by dietary or oral gavage routes.

GLP compliance:

no

Remarks:

generally followed good laboratory practice principles, however no specific study-related Quality Assurance procedures were performed and the report may not contain all of the elements required by GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-8-ol

EC Number:

233-986-8

EC Name:

p-menth-1-en-8-ol

Cas Number:

10482-56-1

Molecular formula:

C10H18O

IUPAC Name:

α,α-4-trimethyl-(1S)-3-cyclohexene-1-methanol

Constituent 2

Reference substance name:

(R)-α,α,4-trimethylcyclohex-3-ene-1-methanol

EC Number:

232-081-5

EC Name:

(R)-α,α,4-trimethylcyclohex-3-ene-1-methanol

Cas Number:

7785-53-7

Molecular formula:

C10H18O

IUPAC Name:

α,α-4-trimethyl-(1R)-3-cyclohexene-1-methanol

Constituent 3

Reference substance name:

1-methyl-4-(1-methylethylidene)cyclohexan-1-ol

EC Number:

209-584-3

EC Name:

1-methyl-4-(1-methylethylidene)cyclohexan-1-ol

Cas Number:

586-81-2

Molecular formula:

C10H18O

IUPAC Name:

1-methyl-4-(1-methylethylidene)-cyclohexanol

impurity 1

Reference substance name:

cis-4-isopropenyl-1-methylcyclohexanol

Cas Number:

7299-41-4

Molecular formula:

C10H18O

IUPAC Name:

cis-4-isopropenyl-1-methylcyclohexanol

impurity 2

Reference substance name:

trans-1-methyl-4-(1-methylethenyl)-cyclohexanol

Cas Number:

7299-40-3

Molecular formula:

C10H18O

IUPAC Name:

trans-1-methyl-4-(1-methylethenyl)-cyclohexanol

impurity 3

Reference substance name:

4-(isopropyl)-1-methylcyclohex-3-en-1-ol

EC Number:

209-585-9

EC Name:

4-(isopropyl)-1-methylcyclohex-3-en-1-ol

Cas Number:

586-82-3

Molecular formula:

C10H18O

IUPAC Name:

4-isopropyl-1-methyl-3-cyclohexen-1-ol

Test material form:

liquid

Details on test material:

Batch No.: 108161
Purity: 83.6% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL MULTICONSTITUENT
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 03 June 2012

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: Approximately 65 days
- Weight at study initiation: 310-350 g
- Housing: Dietary administration - individually housed in polycarbonate or polypropylene cages with stainless steel mesh; Oral gavage administration - housed as five, unless reduced by mortality or isolation.
- Diet (e.g. ad libitum): Rat and Mouse No.1 Maintenance Diet, ad libitum
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 11, 2010 To: September 07, 2010

Administration / exposure

Route of administration:

other: oral (both gavage and dietary)

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Oral gavage formulations were prepared in corn oil weekly or may be in advance of the first day of dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly and may be prepared up to three days in advance of the first day of dosing.
- Mixing appropriate amounts with (Type of food): Rat and Mouse No. 1 Maintenance Diet
- Storage temperature of food: Stored in sealed containers and stored frozen (approximately -20 °C) until issue to the animal room

VEHICLE
- Concentration in vehicle: 750 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Details on mating procedure:

Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first week of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. The samples will be retained frozen (nominally -20 °C) as contingency for any possible future analysis.

Duration of treatment / exposure:

Minimum period: Oral gavage – 2 weeks; Dietary - 2 or 3 weeks

Frequency of treatment:

Dietary administration: continuously; Oral gavage administration: once daily

Details on study schedule:

None

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
7500 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10000 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Five males

Control animals:

no

Details on study design:

- Dose selection rationale: In a standard OECD 422 study (HLS Study No OAD0004), Terpineol multiconstituent has been screened for possible reproductive toxicity. After 5 weeks of dosing at 750 mg/kg/day all males were sterile and subsequent histopathology of the testes and epididymides showed major reductions in the numbers of sperm in the tubules and a high proportion of animals had spermatoceles within the epididymides. No similar effects were detectable at 250 mg/kg/day. It is postulated that the effects may be related to the high peak doses achieved by oral gavage, resulting in atypical metabolism of the test substance. The dietary levels were selected to provide a daily intake similar to the toxic level by oral gavage.

- See table 1 for description of the different groups of animals tested

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals and cages were inspected visually at least twice daily for evidence of ill-health.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded for oral gavage administration. Observations were recorded daily during the first week of treatment and twice weekly during Weeks 2 to 3 (middle and end of each week). Observations were recorded at the following times during the day:
- Pre-dose
- On return of the animal to its home cage
- On completion of dosing of each group
- Between one and two hours after completion of dosing of all groups or, when the duration of dosing is protracted, 1 to 2 hours after completion of each group.
- As late as possible in the working day

BODY WEIGHT:
Bodyweight was recorded on the day that dosing commenced (Week 0), daily (dietary study) or twice weekly (oral gavage study) throughout the dosing and before necropsy

FOOD CONSUMPTION:
Food consumption was recorded daily (dietary study) or twice weekly (oral gavage study). The food supplied to the cage and food spilled was recorded during cage cleaning on measurement weeks. The food remaining in the cages were recorded at the end of measurement periods.

Oestrous cyclicity (parental animals):

Not applicable

Sperm parameters (parental animals):

- Vas deferens (from left side): Sperm sample assessed for motility using a computer assisted sperm analyser (CASA) on all animals of each group. A manual assessment of sperm morphology will be performed on all animals of each group.
- Cauda epididymis (from left side): The cauda epididymis will be weighed and homogenised and the number of sperm will be counted using a computer assisted sperm analyser (CASA) on all animals of each group.
- Testis (from left side): The testis will be homogenised and the number of homogenisation-resistant spermatids will be counted using a computer assisted sperm analyser (CASA) on all animals of each group.

Litter observations:

Not applicable

Postmortem examinations (parental animals):

SACRIFICE:
Animals were sacrificed by carbon dioxide asphyxiation and subsequent exsanguination.

GROSS NECROPSY:
All animals were subjected to a complete macroscopic examination.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 2 were weighed and / or fixed for histopathological examinations.

Postmortem examinations (offspring):

Not applicable

Statistics:

- For categorical data, the proportion of animals was analysed using Fisher’s Exact test.
- For continuous data, Bartlett’s test was applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, groups were compared using either a t-test or Wilcoxon rank sum test.

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs were generally minimal and of the type expected for the age of animals and the duration of the study.
Dosing signs for Group 3 animals receiving oral gavage administration of Terpineol multiconstituent at 750 mg/kg/day, included salivation and chin rubbing. No dosing signs were observed for dietary animals at supplementary oral gavage dosing occasions.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group 1 and 2 animals receiving dietary administration of Terpineol multiconstituent at 7500 or 10000 ppm, showed initial bodyweight loss, between Days 1 and 4 of study, and bodyweight gain thereafter was generally lower than that of animals dosed by gavage.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group 1 and 2 males receiving Terpineol in the diet showed food consumption values lower than expected, particularly at the start of the week 1 suggesting that the test material made the diet less palatable. There was some increase in intake through week one. Males from the 10000 ppm administration group took longer to acclimatise to the diet containing test material however by the end of Week 1 food consumption was approaching normal levels.
On the first day of gavage supplementation (Day 11) a minor decrease in food consumption was recorded for Group 1 and 2 animals, and although food consumption subsequently improved, it never attained the expected levels. This may relate to the nutritive value of corn oil included in the gavage dose.
Group 3 males receiving Terpineol multiconstituent at 750 mg/kg/day, between Days 1-3 of study, had slightly lower than expected food consumption value; this may be associated with the poor bodyweight performance of male 15 within this cage. Food consumption values of Group 3 males receiving Terpineol multiconstituent were within the expected range at all other periods, when the potential effect of the corn oil vehicle is taken into consideration.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The most affected group was Group 3 and there were relatively few histopathological changes in the testes and epididimides of the animals where the bulk of the test material had been given by the dietary route with supplementary gavage dosing. Degenerative changes were seen in the testes: seminiferous tubular atrophy, with associated vacuolation and in the epididymides (reduced sperm numbers and increased numbers of degenerated sperm in the ducts) in Group 3 only.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Week 1 group mean achieved dose level for animals receiving Terpineol multiconstituent at 7500 ppm was 410 mg/kg/day and 10000 ppm was 495 mg/kg/day. These lower than expected results reflected lower than expected food consumption and high initial bodyweight. Consequently additional test material was given by oral gavage (2 x 150 mg/kg/day to Group 1 or 1 x 150 mg/kg/day to Group 2) from Day 11 of dosing. This resulted in overall intake levels of 663 or 678 mg/kg/day for Groups 1 and 2 respectively during the final 11 days of the study. Overall achieved dose level (Days 1-21) for Group 1 animals was 546 mg/kg/day and Group 2 animals was 602 mg/kg/day (see table 3).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility (percentage motile and % progressively motile) was low for all groups of animals. The greatest effect was seen in Group 3. One of 5 males in Group 1 and 4 of 5 males in Group 2 showed essentially normal motility patterns and sperm morphology was largely normal in these animals. The males with poor sperm motility showed high incidences of abnormal sperm, usually decapitate but with increasing incidence of sperm with an abnormal mid-piece in the groups where the whole dose had been given by gavage.
The numbers of sperm in the epididymis were generally within normal ranges for males in Group 2 slightly low in Group 1 but for the majority of animals receiving 750 mg/kg/day of Terpineol multiconstituent, sperm numbers were below expected values. In contrast the numbers of spermatids in the testes of these animals were generally high, especially for the pure material, and slightly high spermatid numbers were seen in both of the groups where diet provided most of the test compound.

Reproductive performance:

not examined

Effect levels (P0)

Dose descriptor:

LOAEL

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Not examined

Effect levels (F1)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 3: Achieved dose - group mean values (mg/kg/day)

Group	Mean 1-7	Mean 1-10	Mean 11-14	Mean 8-14	Mean 15-21	Mean 1-21
Dietary 7500 ppm + supplementary gavage dose 300 mg/kg/day	410	416	368 (668)	566	360 (660)	388 (546)
Dietary 10000 ppm + supplementary gavage dose 150 mg/kg/day	494	519	511 (661)	625	534 (684)	523 (601)

Numbers in parentheses include oral gavage supplementation

Table 4: Sperm analysis - group mean values

Group	Motile sperm (%)	Progressively motile sperm (%)	Cauda epididymis			Testis
			Weight (g)	Sperm count (million/g)	Total (million)	Weight (g)	Sperm count (million/g)	Total (million)
1 (n=5)	19 ± 43	7 ± 16	0.171 ± 0.042	388 ± 248	74 ± 52	1.51 ± 0.56	143 ± 82	248 ± 142
2 (n=5)	78 ± 44	39 ± 23 *	0.211 ± 0.019	761 ± 136 *	162 ± 39 *	1.73 ± 0.22	172 ± 35	295 ± 61
3 (n=5)	8 ± 17	0 ± 0	0.126 ± 0.017	458 ± 184	57 ± 20	0.97 ± 0.08	174 ± 157	176 ± 165

Table 5: Sperm motion data - group mean values

Group

VAP (um/s)

VSL (um/s)

VCL (um/s)

ALH (um/s)

BCF (Hz)

STR (%)

LIN (%)

Elongation (%)

Area (um sq)

Rapid (%)

Medium (%)

Slow (%)

Static (%)

1 (n=5)

30 ± 67

16 ± 35

80 ± 179

5 ± 12

8 ± 18

11 ± 25

4 ± 10

6 ± 13

146 ± 327

13 ± 29

1 ± 1

6 ± 13

81 ± 43

2 (n=5)

126 ± 70

74 ± 42 *

287 ± 164

20 ± 11

28 ± 16

48 ± 27

22 ± 13 *

21 ± 12

618 ± 365

57 ± 33

1 ± 1

20 ± 13

22 ± 44

3 (n=5)

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

8 ± 17

92 ± 17

Table 6: Sperm morphology - group mean values

Group	Total number of sperm examined	Normal (%)	Abnormal (%)	Decapitate (%)	Head abnormal (%)	Neck abnormal (%)	Midpiece abnormal (%)	Tail abnormal (%)
1 (n=4)	809	21.4 ± 42.8	78.6 ± 42.8	77.6 ± 44.8	0.5 ± 1.0	0.4 ± 0.7	31.4 ± 36.3	0.5 ± 0.7
2 (n=5)	1019	78.9 ± 43.8	21.1 ± 43.8	20.1 ± 44.1	0.6 ± 0.4	0.1 ± 0.2	1.3 ± 2.9	0.4 ± 0.4
3 (n=5)	1047	0.1 ± 0.2	99.9 ± 0.2	98.0 ± 2.5	0.1 ± 0.2	1.0 ± 2.1	53.0 ± 43.3	1.0 ± 0.9

Table 7: Organ weights - group mean unadjusted and adjusted values (g) for animals killed at scheduled termination

Group	Terminal bodyweight	Epididymides	Prostate	Seminal Vesicles	Testes
Historical control data	487 ± 34.8	1.165 ± 0.079	1.051 ± 0.207	1.918 ± 0.255	3.526 ± 0.307
1 (n=5)	415 ± 51	1.004 ± 0.197	0.752 ± 0.168	1.533 ± 0.311	2.99 ± 1.16
2 (n=5)	423 ± 42	1.021 ± 0.142	0.894 ± 0.157	1.650 ± 0.261	3.07 ± 0.55
3 (n=5)	420 ± 27	0.771 ± 0.033	0.658 ± 0.087	1.365 ± 0.213	1.96 ± 0.16
	Adjusted means
1 (n=5)		1.013	0.756	1.534	3.04
2 (n=5)		1.012	0.890	1.649	3.01
3 (n=5)		0.770	0.657	1.365	1.96

Historical Control Data, HCD – Rats (IGS CD) males 12.4 - 16.3 weeks of age

Table 8: Macropathology - group distribution of findings for animals killed at scheduled termination

Tissue and finding	Group 1 (n=5)	Group 2 (n=5)	Group 3 (n=5)
Left testis
Blue	1	0	5
Enlarged	0	1	0
Flacid	1	0	5
Small	1	0	5
Left epididymis
Small	1	0	5
Right testis
Blue	1	0	5
Flacid	1	0	5
Small	1	1	5
Right epididymis
Small	1	1	5

Table 9: Histopathology - group distribution of findings for animals killed at scheduled termination

Tissue and finding	Group 1 (n=5)	Group 2 (n=5)	Group 3 (n=5)
Right testis
Seminiferous Tubular Atrophy/Degeneration	1	1	5
Seminiferous Tubular Vacuolation	1	1	4
Spermatid Giant Cells	0	0	5
Right epididymis
Degenerate Spermatogenic Cells in Duct(s)	1	1	5
Epithelial Vacuolation	1	1	0
Inflammation	0	1	1
Reduced Numbers of Spermatozoa	0	1	5
Spermatozoa Absent	1	0	0

Applicant's summary and conclusion

Conclusions:

The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects on the male reproductive system.

Executive summary:

In an investigatory reproductive toxicity study, three groups of Crl:CD(SD) male rats (five/dose) were administered daily with CAS 8000-41-7 by dietary and/or oral gavage routes at the following doses:

- group 1: dietary 7500 ppm + supplementary gavage dose 300 mg/kg/day

- group 2: dietary 10000 ppm + supplementary gavage dose 150 mg/kg/day

- group 3: CAS 8000-41-7 at 750 mg/kg/day by gavage only

During the study, data was recorded on mortality, clinical condition, bodyweight and food consumption. Surviving animals were subjected to a detailed sperm analysis. Testes (L&R), epididymis (L&R), prostate and seminal vesicles were weighed at necropsy and tissues of right testes and epididymis were fixed for histopathological examination.

During week 1 of the study, Group 1 and 2 animals consumed less diet than expected; From Day 11 Group 1 animals, receiving 7500 ppm, also received two daily doses of 150 mg/kg/b.i.d. four to five hours apart and average intake was boosted to 663 mg/kg/day whilst Group 2 animals, receiving 10000 ppm, also received a single daily dose of 150 mg/kg boosting intake to 678 mg/kg/day.

Clinical signs were generally minimal. Dosing signs for Group 3 animals included salivation and chin rubbing. 

Food consumption for Group 1 and 2 animals receiving the test material in the diet was low throughout the study; this was attributed to the palatability of the test material. Bodyweight gains of these animals were also low. Food consumption and bodyweight changes of Group 3 animals were considered to be not adversely affected by the test material. 

Necropsy data indicated that decreases in reproductive organ weights and changes to macroscopic appearance were most marked in the animals receiving Terpineol multiconstituent at 750 mg/kg/day (Table 8). Occasional animals (1/5 and 1/5 in each of the other groups 1 and 2 respectively) also showed changes to testicular and/or epididymal tissue appearances and/or weights (Tables 7 and 8).

Sperm analysis showed that motile sperm with normal morphology were present in 4/5 males of Group 2 and 1/5 males of Group 1 (Tables 4 -6). The outliers in each group were at the extreme of achieved overall exposure for the group suggesting that absolute exposure was important, although the route of exposure and consequently potential to exceed threshold levels was of greater significance.

Microscopic examination indicated there were relatively fewer changes in the testes and epididymides in the animals which were given Terpineol multiconstituent by the dietary route with oral gavage supplementation (Groups 1 and 2), whereas there were significant changes in those which received it solely by oral gavage (Group 3).

The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects.