Juniper, Juniperus mexicana, ext., isomerized, acetylated

Endpoint summary

Description of key information

Skin sensitisation: sensitising (EC3 = 25.7%), female mice, OECD TG 429, 2015

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-02-2015 to 17-06-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J strain, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier (documented in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Microbiological status of animals, when known: No issues reported within the study.
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 19- 24 grams; Body weight variation was within +/- 20% of the sex mean.
- Housing: Group housed, in labelled Makrolon cages (type MIII) sterilised sawdust as bedding material, paper and shelters as cage enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust as cage enrichment.
- Diet: Free access to pelleted rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: None reported.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (18 - 24)
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark
IN-LIFE DATES: From: 04-02-2015 To: to 02-03-2015

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

- Preliminary test: Initially, two test item concentrations were tested; a 25% and 50% concentration. Subsequently, a further test at 100% concentration was treated. The highest concentration chosen was 100%. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). At a 50% test item concentration, ears showed very slight erythema on Days 4 and 5 . Scaliness was observed on the ear(s) of the animals treated at 50% between Days 4 and 6 and for one animal treated at 25% on Day 6 only. At a 100% test item concentration, no erythema (score = 0) and no scaliness was observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values at 25%, 50% and 100%. No mortality occurred and no signs of systemic toxicity were observed in any of the animals examined. The slight body weight losses noted (e.g. -3 g to -1 g) was considered not toxicologically significant since this change was minor in nature and would be considered normal for the species and strain. Based on the pre-screen results, the highest test item concentration selected for the main study was 100% concentration.
- Main test: 0% (vehicle control), 25%, 50% and 100 %. Test concentrations were determined from the results of the preliminary test.

No. of animals per dose:

Preliminary test: Two per concentration: 25%, 50% and 100%.
Main test: Five mice per dose group 0% (vehicle control), 25%, 50% and 100%

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was fully soluble in vehicle [acetone/olive oil (4:1 v/v)]
- Irritation: See below.
- Systemic toxicity: None reported.
- Ear thickness measurements: See below.
- Erythema scores: See below.
- Other: A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6. Initially, two test item concentrations were tested; a 25% and 50% concentration. Subsequently, a further test at 100% concentration was treated. The highest concentration chosen was 100%. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). At a 50% test item concentration, ears showed very slight erythema on Days 4 and 5 . Scaliness was observed on the ear(s) of the animals treated at 50% between Days 4 and 6 and for one animal treated at 25% on Day 6 only. At a 100% test item concentration, no erythema (score = 0) and no scaliness was observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay, females were randomly assigned
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with vehicle (and a further group for the additional dose as control).
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were terminated by intraperitoneal injection (0.2 mL/animal) with Euthasol 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
- Tissue processing and radioactivity measurements: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a scintillation counter. Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
- Mortality/Viability: Twice daily.
- Bodyweights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical Observations: Twice daily (pre- and post-dosing) on Days 1-3. Once daily on Days 4-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Twice daily (pre- and post-dosing) on Days 1-3. Once daily on Days 4-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
- Ear Thickness: Measurements were conducted in the pre-screen test and in the main study. A digital thickness gauge was used to measure the ear thickness of each ear prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6 in order to monitor for any changes in ear thickness.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Analysis was conducted according to Basketter DA et al., A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.

Positive control results:

In a separate 'positive control study' performed according to OECD TG 429 during November 2014, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde. The positive control was tested at concentrations 5%, 10 %and 25% in Acetone/Olive oil (4:1 v/v). The highest concentration tested showed a Stimulation Index (SI) of 3.7 ± 1.1 (at 25% v/v) and met the criteria for a 'positive' result. An EC3 value of 9.8% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 14.4, 16.5, 14.5, 13.4, 14.1 and 17.3%

Parameter:

EC3

Remarks:

%

Value:

25.7

Remarks on result:

other:

Remarks:

see table below

Parameter:

SI

Remarks:

mean (n=5)

Value:

2.8

Variability:

± 0.7

Test group / Remarks:

25% in acetone:olive oil (4:1)

Parameter:

SI

Remarks:

mean (n=5)

Value:

9.8

Variability:

± 2.6

Test group / Remarks:

50% in acetone:olive oil (4:1)

Parameter:

SI

Remarks:

mean (n=5)

Value:

32.6

Variability:

± 6.4

Test group / Remarks:

100%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The EC3 was determined based on the linear interpolation. The mean values and standard deviations were calculated in the body weight tables in the study report. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
EC3 = 25.71%
Where: a = 25, b = 2.8, c = 50 and d = 9.8

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. A single slight body weight loss (female #17) noted at 100% was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent. This would be considered normal for the species and strain.

Preliminary screening test:

No irritation or signs of systemic toxicity were observed except for the very slight erythema on the right ear treated at 50% on days 4 and 5. Scaliness was observed on the ear(s) treated at 50% between days 4 and 6 and in one incident at 25% at day 6 only. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test item concentration selected for the main study was a 100% concentration.

 

Main test:

No irritation of the ears was observed except for very slight erythema observed at 100% on day 3. Variations on ear thickness during the observation period were less than 25% from day 1 pre-dose values. No mortality occurred and there were no clinical signs of systemic toxicity. Body weights and body weight gain remained in the same range as the controls over the study period. A single slight body weight loss (female #17) noted at 100% was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent. This would be considered normal for the species and strain. The auricular lymph nodes of the vehicle control group and 25% group were considered normal in size. The nodes of the 50% and 100% groups were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the females. The radioactive disintegrations per minute (dpm) and stimulation index (SI) are given in the table below. The SI values calculated for the substance concentrations 25%, 50% and 100% were 2.8, 9.8 and 32.6, respectively. These results indicate that the test item could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 25.7 % was calculated.

Table 1. Results from the definitive test

Group	TS (%) #1	Number	Size nodes #2		DPM / animal #3	mean			mean
			left	right		DPM ± SEM #4			SI ± SEM
1	0	1	n	n	306	315	±	51	1.0	±	0.2
		2	n	n	270
		3	n	n	188
		4	n	n	314
		5	n	n	498
2	25	6	n	n	1042	885	±	186	2.8	±	0.7
		7	n	n	1540
		8	n	n	715
		9	n	n	530
		10	n	n	596
3	50	11	+	+	1338	3091	±	651	9.8	±	2.6
		12	+	+	2686
		13	+	+	2658
		14	+	+	3465
		15	+	+	5307
4	100	16	+	+	9094	10277	±	1156	32.6	±	6.4
		17	+	+	7557
		18	+	+	13677
		19	+	+	12297
		20	+	+	8760

#1. TS = test substance (% w/w).

#2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

#3. DPM = Disintegrations per minute

#4. SEM = Standard Error of the Mean

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is considered to be sensitising to skin with EC3 of 25.7%.

Executive summary:

The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600 guidelines under GLP to assess the skin sensitisation potential of the test item in the CBA/J strain mouse following topical application to the dorsal surface of the ear. In a preliminary screening test mice were treated by daily application of 25 μl of the test substance at 25%, 50% in acetone/olive oil 4:1 diluted test item and subsequently 100% v/v to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice was observed twice daily and local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6. A mean ear thickness increase of equal to or greater than 25% and/or well-defined irritation at the most (maximum grade 2) was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. No irritation or signs of systemic toxicity were observed except for the very slight erythema on the right ear treated at 50% on days 4 and 5. Scaliness was observed on the ear(s) treated at 50% between days 4 and 6 and in one incident at 25% at day 6 only. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% and 100% v/v. In the main test, three groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). No irritation of the ears was observed in the animals except for the very slight erythema observed for the animals treated at 100% on Day 3. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Body weights were within the range seen for historic control animals. The auricular lymph nodes of the animals of the control group and 25% Group were considered normal in size. The lymph nodes of the animals treated at 50% and 100% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 885, 3091 and 10277 DPM, respectively. The mean DPM/animal value for the vehicle control group was 315 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 2.8, 9.8 and 32.6, respectively. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 25.7 % was calculated. Under the conditions of this study, the test item would be considered to be classified as skin sensitizer (category 1B) under Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study : OECD TG 429, 2015 : The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600 guidelines under GLP to assess the skin sensitisation potential of the test item in the CBA/J strain mouse following topical application to the dorsal surface of the ear. In a preliminary screening test mice were treated by daily application of 25 μl of the test substance at 25%, 50% in acetone/olive oil 4:1 diluted test item and subsequently 100% v/v to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice was observed twice daily and local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6. A mean ear thickness increase of equal to or greater than 25% and/or well-defined irritation at the most (maximum grade 2) was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. No irritation or signs of systemic toxicity were observed except for the very slight erythema on the right ear treated at 50% on days 4 and 5. Scaliness was observed on the ear(s) treated at 50% between days 4 and 6 and in one incident at 25% at day 6 only. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% and 100% v/v. In the main test, three groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). No irritation of the ears was observed in the animals except for the very slight erythema observed for the animals treated at 100% on Day 3. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Body weights were within the range seen for historic control animals. The auricular lymph nodes of the animals of the control group and 25% Group were considered normal in size. The lymph nodes of the animals treated at 50% and 100% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 885, 3091 and 10277 DPM, respectively. The mean DPM/animal value for the vehicle control group was 315 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 2.8, 9.8 and 32.6, respectively. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 25.7 % was calculated. Under the conditions of this study, the test item would be considered to be classified as skin sensitizer (category 1B) under Regulation (EC) No 1272/2008.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B.

 

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.