Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an FDA-sponsored study (FDA, 1973), reported in the Flavour and Fragrance HPV Consortium (FFHPVC) Test Plan for Bicyclic Terpene Hydrocarbons, pregnant female Wistar rats, CD-1 mice and golden hamsters were dosed by gavage with up to 260, 560 and 600 mg/kg/day FDA 71-28 in corn oil respectively, on gestation days 6 to 15. There was no evidence of reproductive toxicity in any species up to and including the highest dose tested, 260, 560 and 600 mg/kg/day respectively. The study pre-dates GLP and was not consistent with current guideline requirements for reproductive toxicity.

The most abundant constituents of TOPP are represented by existing reproductive toxicity data and information that is available on minor constituents indicates that significant reproductive effects are not expected for these types of chemical structures. The registrants therefore consider that on the grounds of animal welfare and minimising in vivo studies in vertebrates, the conduct of further studies is not warranted for this endpoint. Existing data are adequate to reach an appropriate hazard conclusion for reproductive toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Only pregnant females were treated. The original study was not available for review.
Qualifier:
no guideline followed
Principles of method if other than guideline:
US Food and Drug Administration-sponsored study to investigate reproductive and developmental toxicity in rats, mice and hamsters. Test substance was administered to pregnant females only.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Test animals were individually housed in mess-bottom cages in a temperature and humidity-controlled room.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
No details available.
Details on mating procedure:
Adult female Wistar rats were mated with untreated young adult males and observation of vaginal sperm plugs was considered day 0 of gestation.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Day 6 to Day 15 of gestation
Frequency of treatment:
Once per day
Details on study schedule:
No details available
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
56 mg/kg bw/day (actual dose received)
Dose / conc.:
260 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22-23 per dose
Control animals:
yes, concurrent vehicle
Details on study design:
No details available.
Positive control:
Yes. 250 mg/kg/day aspirin.
Parental animals: Observations and examinations:
Maternal body weights were recorded on days 0, 6, 11, 15, and 17 of gestation. Females were observed daily for appearance and behavior. Food consumption and body weight were monitored to eliminate any abnormalities that may be associated with anorexia in pregnant females.

On Day 17 all dams were subjected to Caesarian section and the number of implantation sites, resorption sites, live fetuses, dead fetuses, and body weight of live pups were recorded.

Gestation index, mortality, number of implantation sites, number of corpora lutea, litter size and weights, sex and sex ratio of pups, and gross abnormalities to pups were reported. One-third of fetuses of each litter underwent detailed visceral examination at 10x magnification. The remaining two-thirds were stained with alizarin red S dye/KOH and examined for skeletal defects.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
On Day 17 all dams were subjected to Caesarian section and the number of implantation sites, resorption sites, live fetuses, dead fetuses, and body weight of live pups were recorded.

Gestation index, mortality, number of implantation sites, number of corpora lutea, litter size and weights, sex and sex ratio of pups, and gross abnormalities to pups were reported.
Postmortem examinations (parental animals):
The urogenital tract of each dam was examined for anatomical abnormalities.
Postmortem examinations (offspring):
One-third of fetuses of each litter underwent detailed visceral examination at 10x magnification. The remaining two-thirds were stained with alizarin red S dye/KOH and examined for skeletal defects.
Statistics:
None
Reproductive indices:
No information
Offspring viability indices:
No information
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
The administration of up to and including 260 mg/kg bw/day of test article FDA 71-28 to pregnant rats on days 6 through 15 of gestation had no
effects on nidation, reproduction, or maternal survival.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 260 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: No adverse effects observed
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
The administration of up to and including 260 mg/kg bw/day of test article FDA 71-28 to pregnant rats on days 6 through 15 of gestation had no
effects on any measured foetal parameter.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 260 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed
Reproductive effects observed:
no
Conclusions:
In a limited summary of a one-generation reproduction study with Wistar rats, there was no evidence of reproductive toxicity up to and including the highest dose tested, 260 mg/kg/day. The study pre-dates GLP and was not consistent with current guideline requirements for reproductive toxicity.
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Only pregnant females were treated. The original study was not available for review.
Qualifier:
no guideline followed
Principles of method if other than guideline:
US Food and Drug Administration-sponsored study to investigate reproductive and developmental toxicity in rats, mice and hamsters. Test substance was administered to pregnant females only.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
female
Details on test animals and environmental conditions:
Test animals were individually housed in mess-bottom cages in a temperature and humidity-controlled room.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
No details available.
Details on mating procedure:
No information
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Day 6 to Day 15 of gestation
Frequency of treatment:
Once per day
Details on study schedule:
No details available
Dose / conc.:
6 mg/kg bw/day (actual dose received)
Dose / conc.:
26 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
560 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20-21 per dose
Control animals:
yes, concurrent vehicle
Details on study design:
No details available.
Positive control:
Yes. 150 mg/kg/day aspirin.
Parental animals: Observations and examinations:
Maternal body weights were recorded on days 0, 6, 11, 15, and 17 of gestation. Females were observed daily for appearance and behavior. Food consumption and body weight were monitored to eliminate any abnormalities that may be associated with anorexia in pregnant females.

On Day 17 all dams were subjected to Caesarian section and the number of implantation sites, resorption sites, live fetuses, dead fetuses, and body weight of live pups were recorded.

Gestation index, mortality, number of implantation sites, number of corpora lutea, litter size and weights, sex and sex ratio of pups, and gross abnormalities to pups were reported. One-third of fetuses of each litter underwent detailed visceral examination at 10x magnification. The remaining two-thirds were stained with alizarin red S dye/KOH and examined for skeletal defects.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
On Day 17 all dams were subjected to Caesarian section and the number of implantation sites, resorption sites, live fetuses, dead fetuses, and body weight of live pups were recorded.

Gestation index, mortality, number of implantation sites, number of corpora lutea, litter size and weights, sex and sex ratio of pups, and gross abnormalities to pups were reported.
Postmortem examinations (parental animals):
The urogenital tract of each dam was examined for anatomical abnormalities.
Postmortem examinations (offspring):
One-third of fetuses of each litter underwent detailed visceral examination at 10x magnification. The remaining two-thirds were stained with alizarin red S dye/KOH and examined for skeletal defects.
Statistics:
None
Reproductive indices:
No information
Offspring viability indices:
No information
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
The administration of up to and including 560 mg/kg bw/day of test article FDA 71-28 to pregnant mice on days 6 through 15 of gestation had no effects on nidation, reproduction or maternal survival. The number and types of abnormalities seen in tissues of the dam or pups of the test groups did not differ for the number and type occurring spontaneously in the positive or negative controls.
Dose descriptor:
NOAEL
Effect level:
>= 560 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: No adverse effects observed
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
The administration of up to and including 560 mg/kg bw/day of test article FDA 71-28 to pregnant mice on days 6 through 15 of gestation had no effects on any measured fetal parameter. The number and types of abnormalities seen in tissues of the dam or pups of the test groups did not differ for the number and type occurring spontaneously in the positive or negative controls.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 560 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed
Reproductive effects observed:
no
Conclusions:
In a limited summary of a one-generation reproduction study with CD-1 mice, there was no evidence of reproductive toxicity up to and including the highest dose tested, 560 mg/kg/day. The study pre-dates GLP and was not consistent with current guideline requirements for reproductive toxicity.
Effect on fertility: via oral route
Dose descriptor:
NOAEL
260 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No reproductive toxicity studies are available for TOPP. Limited data are, however, available for an essential oil (oil of nutmeg, FDA 71 -28), which consists predominantly (80-90%) of bicyclic terpene C10H16hydrocarbons {α-pinene (20-25%), β-pinene (15-18%) and sabinene (38-42%)}, and is thus representative of the major constituents of TOPP.

α-Pinene comprises between 10 and 85% by weight of TOPP, and β-pinene 0-40%. Other principal constituents of TOPP are δ-3-carene (0 - 40%) and dipentene (0 - 20%). Lower concentrations (up to around 5% each) of other terpenes are present. These terpene constituents are also present in varying proportions in other forms of turpentine, all of which are captured in the general description of CAS Number 8006-64-2.

Relevant data for a number of other constituents, including the sulfur-containing constituents, are also available and these are also taken into consideration in order to reach an appropriate overall hazard conclusion for reproductive toxicity of TOPP.

As described in Section 1.3 of the Chemical Safety Report, constituents of TOPP can be grouped into ‘blocks’ according to chemical structure and properties and these are shown in Table 5.10.3 below along with a summary of available reproductive toxicity data for each block.

Table 5.10.3 Available reproductive toxicity data for TOPP Blocks

Block number

Block name

Typical %wt in TOPP

Representative constituents with reproductive toxicity data

1

Pinene Block

71.5

Oil of nutmeg

2

Delta-3-carene Block

13

No data available

3

Dipentene Block

7.8

Terpinolene

4

Dimethyl sulfide Block

1.2

No data available

5

Methyl mercaptan Block

0.06

No data available

6

Sesquiterpenes Block

4.3

No data available

7

Terpene alcohols Block

1.0

Terpineol multi
Anethole

8

Camphene Block

0.81

Camphene

9

Dimethyl disulfide Block

0.32

Dimethyl disulfide

 

Oil of nutmeg

In an FDA-sponsored study (FDA, 1973), reported in the Flavour and Fragrance HPV Consortium (FFHPVC) Test Plan for Bicyclic Terpene Hydrocarbons, pregnant female Wistar rats, CD-1 mice and golden hamsters were dosed by gavage with up to 260, 560 and 600 mg/kg/day FDA 71-28 in corn oil respectively, on gestation days 6 to 15. There was no evidence of reproductive toxicity in any species up to and including the highest dose tested, 260, 560 and 600 mg/kg/day respectively. The study pre-dates GLP and was not consistent with current guideline requirements for reproductive toxicity.

Terpinolene/Iso terpinolene (CAS 586-62-9)

Terpinolene and isoterpinolene (both assigned CAS number 586-62-9) are each present in TOPP at concentrations in the range 0 – 5% by weight. In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), these constituents are members of Block 3 (Dipentene block) which in total typically represents approximately 7% by weight of the registration substance.In a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test conducted according to OECD Test Guideline No 422 and in compliance with GLP, three groups of Sprague-Dawley Crl: CD®BR strain rats, each comprising of ten males and ten females for the main phase (except for control and top dose: 5 males/dose) received terpinolene monoconstituent at doses of 800, 2500 or 5000 ppm by dietary admixture (initially mixed with 2% corn oil) (Harlan Laboratories, 2014a). Main phase males were dosed daily during premating and mating periods and up to 42 days and females were dosed up to 63 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females). During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption, haematology, blood chemistry, oestrous cycle, mating performance, fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. Effects relevant to systemic toxicity are described in Section 7.5.

Reduced litter weights were evident for females treated with 5000 ppm on Day 7 post partum when compared to controls. Mean offspring weights were reduced from these litters on Day 7 post partum resulting in a reduction in body weight gain between Days 4 and 7 post partum.

In the absence of any effect in litter size or litter viability the intergroup differences detected in offspring development were considered to be related to the reduced food consumption of the adult females, which caused reduced body weight in maternal animals rather than a direct toxic effect on the offspring. No treatment-related effects were detected in mating performance, fertility and gestation lengths:

- all animals mated within the first five days of pairing;

- there were no differences in conception rates for treated animals;

- the distribution of gestation lengths for treated females was comparable to controls. The majority of females showed gestation lengths between 21 1/2 and 23 days.

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) of terpinolene monoconstituent for systemic toxicity for females and males was 5000 ppm, (corresponding to 298 and 289 mg/kg bw/day, respectively), the NOAEL for reproductive toxicity was 5000 ppm (corresponding to 294.6 mg/kg bw/day) and the NOAEL for maternal toxicity and developmental toxicity was 5000 ppm (corresponding to 371mg/kg bw/day).

Terpineol multi (CAS 8000-41-7)

In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), terpene alcohol constituents are members of Block 7 which in total typically represents approximately 1% by weight of the registration substance. The test substance Terpineol Multi (CAS 8000-41-7) is a multiconstituent substance containing predominantly α-terpineol (CAS 98-55-5; 0 – 5% by weight of TOPP) with smaller amounts of β-terpineols, γ-terpineol, δ-terpineol, terpinen-1-ol-4, terpinen-3-ol-1, borneol, isoborneol, fenchol and other minor constituents. Please refer to the confidential test material information in the IUCLID dataset for further details on the composition of the test material (Huntingdon Life Sciences, 2010).

Terpineol multi (terpineol; CAS 8000-41-7) has been tested in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test, conducted following GLP guidelines and OECD Test Guideline 422. Three groups, each comprising of ten male and ten female rats for the main (reproductive) phase and five female rats for the toxicity phase received terpineol multi by gavage at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 ml/kg bw/day. The systemic effects observed during the study are discussed in Section 5.6.The most significant finding was changes in relative and absolute testis and epididymal weights in high dose males. Gross necroscopy showed a range of testicular findings including small, blue and flaccid testes. Microscopic examination revealed reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides of males receiving 750 mg/kg bw/day. The effects observed in male reproductive organis persisted until the end of the recovery period. In the females mated with these males, fewer sperm were detected in vaginal smears and no pregnancies resulted in females. It was considered that the testicular and epididymal effects observed in males receiving 750 mg/kg bw/day would have been sufficient to prevent fertilisation and it was assumed, therefore, that females receiving 750 mg/kg bw/day have not been tested for female-specific reproductive effects. There were no effects on oestrous cycles, pre-coital interval, mating performance and fertility up to 250 mg/kg bw/day. It was concluded that the No-Observed-Adverse–Effect-Level (NOAEL) for males and females was 250 mg/kg bw/day and ≥250 mg/kg bw/day respectively.

Trans-Anethole (CAS 4180-23-8)

Trans-anethole (CAS 4180-23-8) may be present in TOPP at up to 1% by weight and is another representative of Block 7 (terpene alcohols). The disseminated REACH dossier for anethole reports two reproductive toxicity studies.In a one-generation reproductive toxicity study with Wistar rats, no treatment related effects were reported for either the parents or offspring. The NOAEL is considered to be between 600 and 1500 mg/kg (bw).

In a two-generation reproductive toxicity study, similar to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study) with anethole, no treatment related effects were observed and the NOAEL is considered to be between 600 and 1500 mg/kg (bw)(ECHA 2013c).

Camphene (CAS 79-92-5)

Camphene is present in TOPP at 0 – 5% by weight and represents Block 8 (camphene). A 28-day repeated dose oral toxicity study, conducted according to OECD 407, is reported in the disseminated REACH dossier[1]for this substance, with a conclusion of NOELs of 250 mg/kg  bw/day for female rats, and <62.5 mg/kg  bw/day for males. However, the effects described in the disseminated dossier are male rat-specific changes in kidneys, which are of no toxicological importance or relevance to man, and increased absolute and relative liverweights, which might be a result of adaptive changes. However, it is considered that the NOAEL to be used in risk assessment is 250 mg/kg bw/day. No effects were reported on reproductive organs.

Dimethyl disulfide (CAS 624-92-0)

The disseminated REACH dossier for dimethyl disulfide reports a key 2-generation reproductive toxicity study in rabbits, conducted according to OECD 416 by the inhalation route. There were no effects on reproductive or developmental parameters up to the highest dose tested, 80 ppm.


[1] Accessed 8thNovember 2016

Effects on developmental toxicity

Description of key information

In an FDA-sponsored study (FDA, 1973), reported in the Flavour and Fragrance HPV Consortium (FFHPVC) Test Plan for Bicyclic Terpene Hydrocarbons, pregnant female Wistar rats, CD-1 mice and golden hamsters were dosed by gavage with up to 260, 560 and 600 mg/kg/day FDA 71-28 in corn oil on gestation days 6 to 15. There was no evidence of developmental toxicity in any species up to and including the highest dose tested, 260, 560 and 600 mg/kg/day respectively.

An oral (gavage) developmental toxicity study (LPT, 1992) conducted largely according to OECD 414 and with GLP involved administration of camphene (technical, 78%) to pregnant rats on gestation days 6-15. The study identified maternal and developmental NOELs at 250 mg/kg bw/day. The NOAEL for maternal effects was 250 mg/kg bw/day and for foetal effects >= 1000 mg/kg bw/day.

The most abundant constituents of TOPP are represented by existing developmental toxicity data and information that is available on minor constituents indicates that developmental effects are not expected for these types of chemical structures. The registrants therefore consider that on the grounds of animal welfare and minimisingin vivostudies in vertebrates, the conduct of further studies is not warranted for this endpoint. Existing data are adequate to reach an appropriate hazard conclusion for developmental toxicity.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21.04.1992 to 06.10.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only two dose levels were tested
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Only two dose levels were tested)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, HAGEMANN GmbH, D-4923 Extertal 1
- Age at study initiation: ca. 54 days old
- Weight at study initiation: 180 - 190 g
- Housing: The dams were kept singly in MAKROLON cages type III.
- Diet (e.g. ad libitum): ALTROMIN 1314 (supplied by: ALTROMIN GmbH, P.O.Box 285, D-4937 Lage/Lippe) served as food, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: Seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 50 ± 15 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 28.04.1992 To: 05.08.1992
Route of administration:
oral: gavage
Vehicle:
other: sesame oil, DAB 10
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance-vehicle mixtures were freshly prepared each day immediately before dosing.
Volume of administration: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the concentration in the substance-vehicle preparations, 2 samples of 10 mL (each one for the concentrations 50.0 mg/Camphene/mL suspension and 200.0 mg Camphene/mL suspension) were analyzed at the start and at the end of treatment.
The determination of the concentrations was performed by gaschromatography using a FID detector.
Details on mating procedure:
Fertile ('proved') 4 - 12 months old male rats of the same breed served as partners. They were repeatedly employed, at the earliest three days after successful copulation. The female breeding partners were randomly chosen.
Matings were monogamous.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From the 6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
20 days (from day 0 to day 20 of pregnancy)
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
20 pregnant female rats per group (plus 15 reserve animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
During a pilot study using 3 pregnant rats from the same strain as selected for the main experiment, a Camphene dose-Ievel of 1000 mg/kg b.w., by gavage, was administered from day 6 to 15 of pregnancy. 1000 mg/kg b.w. is the highest requested dose according to the
OECD method 414 (limit test for embryotoxicity).
Except for a slight transient reaction after the first dosing, 1000 mg/kg b.w. was well-tolerated by the dams. 1000 mg/kg b.w. did not influence the prenatal development. Based on the afore-described results, 250 mg and 1000 mg Camphene/kg b.w./day, by gavage, treatment from the 6th to 15th day of prenancy were selected by the sponsor as doses for the main experiment.
Maternal examinations:
Daily checks (once in the morning and once as late on the day as practicable) were performed on behaviour, external appearance, mortality and faeces.
Body weight was ascertained daily - always at the same time in the morning - and these measurements were also used for calculating the daily amount of test compound to be administered.
Body weight change: The difference between the body weight on the day of weighing and the body weight on the day -4 weighing was calculated.
Food consumption was determined daily by weighing the residue. Intake of drinking-water was observed daily.
Ovaries and uterine content:
On the 20th day of gestation the surviving rats were laparotomised under ether narcosis. The ovaries and uteri were removed and examined. To check for possible drug effects a dissection was carried out which included macroscopic examination of internal organs. A full macroscopic inspection was carried out in the prematurely deceased dams as soon as possible after their exitus.
Fetuses were removed and the following examinations performed:
A count was taken of fetuses and placentae.
Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
Number and size of resorptions were determined.
Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
The uterus weight was determined (with and without fetuses), weight of the ovaries was determined.
Weights and length of fetuses and weight of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the
mean litter weight).
Fetal examinations:
Fetuses were inspected externally for damages, especially for malformations.
Fetuses were dissected and the number and type of possible variations (incl. retardations) or malformations was determined macroscopically:
In 50% of the number of fetuses/L thorax and peritoneal cavity (without damage to ribs and sternum) were opened; location, size, and condition of the
internal organs were determined. Then the skeletons were stained with Alizarin according to DAWSON, the skeletal system was examined. Determination of the number and an examination of retardations, variations as well as malformations was carried out.
In 50% of the number of fetuses/L body sections were made and examined for variations according to WILSON.
Statistics:
The comparison of malformation and variation rates was carried out using R.A.FISHER's exact test (p < = 0.05). All other data were evaluated in the following way:
Homogeneity of variances were tested by the Bartlett chi-square test, and - if the variances were homogeneous - a one-way analysis of variance was applied. When the results indicated a significant difference among groups the DUNNETT test (p <= 0.01) was used to compare the experimental groups with the control group.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No substance-related mortality was observed. Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs. No clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range, body weight gain showed no influence of the test compound, also. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg b.w.) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No substance-related pathological changes were detected at autopsy.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
1000 mg Camphene/kg b.w./day caused a slight but not significant (at p < = 0.01) increase of the resorption rate and - consequently - of the post-implantation loss (resorption rate and post-implantation loss substance-treated group: 11.5%, control group: 5.2%). No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue (WILSON's method) and skeletal examination (staining of the skeleton according to DAWSON's method) revealed no substance-related variations and/or retardations. One malformed fetus at 1000 mg/kg (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail). No dead fetuses occurred in the substance treated and control dams.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
no
Under the present test conditions, the NOEL for the dams and for the 
fetal organism was 250 mg Camphene/kg bw/day. 
Camphene did not possess teratogenic properties.
Conclusions:
Under the present test conditions, the NOEL for the dams and for the fetal organism was 250 mg Camphene/kg bw/day.
Camphene did not possess teratogenic properties.
Executive summary:

Pregnant Sprague-Dawley rats were orally treated with the test substance by gavage during the 'critical' phase of organogenesis (daily from the 6th to 15th day of pregnancy). Test method was according to OECD guideline 414. Tested concentrations were 0, 250 and 1000 mg/kg bw/day, using sesame oil as vehicle. 20 pregnant rats were used in each group. Their development during the gestation period is observed. On day 20 of pregnancy the animals were laparotomised and examined macroscopically for implantation sites, resorptions in the uterus and for condition of the fetuses.

Influence on the dam:

No substance-related mortality was observed. Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs. No clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range, body weight gain showed no influence of the test compound, also. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No substance-related pathological changes were detected at autopsy.

Influence on the fetus:

1000 mg Camphene/kg bw/day caused a slight but not significant (at p < = 0.01) increase of the resorption rate and - consequently - of the post-implantation loss (resorption rate and post-implantation loss substance-treated group: 11.5%, control group: 5.2%). No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue (WILSON's method) and skeletal examination (staining of the skeleton according to DAWSON's method) revealed no substance-related variations and/or retardations. One malformed fetus at 1000 mg/kg bw/day (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail). No dead fetuses occurred in the substance treated and control dams.

Under the present test conditions, the NOEL for the dams and for the fetal organism was 250 mg Camphene/kg bw/day. Camphene did not possess teratogenic properties.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

No developmental toxicity data are available for TOPP. Limited data are, however, available for an essential oil (oil of nutmeg, FDA 71-28), which consists predominantly (80-90%) of bicyclic terpene C10H16hydrocarbons {α-pinene (20-25%), β-pinene (15-18%) and sabinene (38-42%), and is thus representative of the major constituents of TOPP.

α-Pinene comprises between 10 and 85% by weight of TOPP, and β-pinene 0-40%. Other principal constituents of TOPP are δ-3-carene (0 - 40%) and dipentene (0 - 20%). Lower concentrations (up to around 5% each) of other terpenes are present. These terpene constituents are also present in varying proportions in other forms of turpentine, all of which are captured in the general description of CAS Number 8006-64-2.

Relevant data for a number of constituents, including the sulfur-containing constituents, are also available and these are also taken into consideration in order to reach an appropriate overall hazard conclusion for developmental toxicity of TOPP.

As described in CSR Section 1.3, constituents of TOPP can be grouped into ‘blocks’ according to chemical structure and properties and these are shown in Table 5.10.4 below along with a summary of available developmental toxicity data for each block.

Table 5.10.4 Available developmental toxicity data for TOPP Blocks

 

Block number

Block name

Typical %wt in TOPP

Representative constituents with developmental toxicity data

1

Pinene Block

71.5

Oil of nutmeg

2

Delta-3-carene Block

13

No data available

3

Dipentene Block

7.8

Terpinolene

4

Dimethyl sulfide Block

1.2

No data available

5

Methyl mercaptan Block

0.06

No data available

6

Sesquiterpes Block

4.3

No data available

7

Terpene alcohols Block

1.0

Terpineol multi
Anethole

8

Camphene Block

0.81

Camphene

9

Dimethyl disulfide Block

0.32

Dimethyl disulfide

Oil of nutmeg

In an FDA-sponsored study (FDA, 1973), reported in the Flavour and Fragrance HPV Consortium (FFHPVC)Test Plan for Bicyclic Terpene Hydrocarbons, pregnant female Wistar rats, CD-1 mice and golden hamsters were dosed by gavage with up to 260, 560 and 600 mg/kg/day FDA 71-28 in corn oil on gestation days 6 to 15. There was no evidence of developmental toxicity in any species up to and including the highest dose tested, 260, 560 and 600 mg/kg/day respectively. The study pre-dates GLP and was not consistent with current guideline requirements for reproductive toxicity.

Camphene (CAS 79-92-5)

Camphene is present in TOPP at 0 – 5% by weight and represents Block 8 (camphene). An oral (gavage) developmental toxicity study (Mitterer et al., 1992) conducted largely according to OECD 414 and with GLP involved administration of camphene (technical, 78%) to pregnant rats on gestation days 6-15. The study identified maternal and developmental NOELs at 250 mg/kg bw/day. The NOAEL for maternal effects was 250 mg/kg bw/day and for foetal effects ≥1000 mg/kg bw/day. Effects at 1000 mg/kg bw/day included transient reduced motility and salivation in dams, and slightly (though not statistically significantly) increased early resorption and post-implantation loss (5.2%, 6.9%, 11.5% in the control, 250 mg/kg bw/day and 1000 mg/kg bw/day groups, respectively). No teratogenic effects were detected. Although camphene is a minor constituent of TOPP (up to 2% by weight), it is a bicyclic terpene hydrocarbon and structural isomer of α- and β-pinene, which are the major constituents.

Terpinolene/Iso terpinolene (CAS 586-62-9)

Terpinolene and isoterpinolene (both assigned CAS number 586-62-9) are each present in TOPP at concentrations in the range 0 – 5% by weight. In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), these constituents are members of Block 3 (Dipentene block) which in total typically represents approximately 7% by weight of the registration substance. In a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test (Harlan Laboratories, 2014a) conducted according to OECD Test Guideline No 422 and in compliance with GLP, three groups of Sprague-Dawley Crl: CD®BR strain rats, each comprising of ten males and ten females for the main phase (except for control and top dose: 5 males/dose), five females for the toxicity phase and 5 males and 5 females/dose (control and top dose) for the recovery phase received high purity terpinolene monoconstituent at dietary concentrations of 0, 800, 2500 or 5000 ppm (initially mixed with 2% corn oil). Main phase males and toxicity phase females were dosed daily for 42 days. Recovery phase animals were treated with the high dose or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. During the study, data was recorded on clinical condition and systemic effects, reproductive performance, clinical signs of offspring and litter sizes and weights. The systemic effects observed during the study are discussed in Section 5.6. No treatment related effects were detected in litter size and viability. There were no test-substance related necropsy findings in either interim deaths or scheduled kill offspring. It was concluded in the study report that the NOAEL for developmental toxicity was 2500 ppm, however as no adverse effects were reported, it is concluded by the reviewer that the NOAEL for developmental toxicity is 5000 ppm, equivalent to371 mg/kg bw/day (females, days 0-6 of gestation).

Terpineol multi (CAS 8000-41-7)

In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), terpene alcohol constituents are members of Block 7 which in total typically represents approximately 1% by weight of the registration substance. The test substance Terpineol Multi (CAS 8000-41-4) is a multiconstituent substance containing predominantly α-terpineol (CAS 98-55-5; 0 – 5% by weight of TOPP) with smaller amounts of β-terpineols, γ-terpineol, δ-terpineol, terpinen-1-ol-4, terpinen-3-ol-1, borneol, isoborneol, fenchol and other minor constituents. Please refer to the confidential test material information in the IUCLID dataset for further details on the composition of the test material (Huntingdon Life Sciences, 2010).

Terpineol multi (terpineol; CAS 8000-41-7) has been tested in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test, conducted following GLP guidelines and OECD Test Guideline 422. Three groups, each comprising of ten male and ten female rats for the main (reproductive) phase and five female rats for the toxicity phase received terpineol multi by gavage at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 ml/kg bw/day. The systemic effects observed during the study are discussed in Section 5.6. Testicular and epididymal effects were observed in males receiving 750 mg/kg/day, and no pregnancies resulted from matings with these animals, therefore developmental effects were assessed only at 60 and 250 mg/kg bw/day. There were no adverse effects on survival, growth and development of offspring up to Day 7 of age up to 250 mg/kg bw/day. It was concluded that the NOAEL for developmental toxicity was ≥250 mg/kg bw/day.

Trans-Anethole (CAS 4180-23-8)

Trans-anethole (CAS 4180-23-8) may be present in TOPP at up to 1% by weight and is another representative of Block 7 (terpene alcohols). The disseminated REACH dossier for anethole reports a prenatal developmental toxicity study (similar to OECD Guideline 414). The NOAEL for maternal toxicity was set as 35 mg/kg (bw) and the NOAEL for developmental effects as 175 mg/kg (bw). However, the test substance did not cause any developmental effects on the rat foetus at doses below those causing maternal toxicity (reduced body weight and feed consumption).

Dimethyl disulfide (CAS 624-92-0)

The disseminated REACH dossier for dimethyl disulfide reports a key pre-natal developmental toxicity study, conducted according to OECD 414 by the inhalation route. The NOAEL for developmental effects was 135 ppm, the highest dose tested. In addition, a 2-generation reproductive toxicity study was conducted according to OECD 416 by the inhalation route. There were no effects on reproductive or developmental parameters up to the highest dose tested, 80 ppm.

Justification for classification or non-classification

None of the repeated dose toxicity studies with constituents of TOPP showed effects that were of significance for classification for reproductive or developmental toxicity.