Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-27 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on June 17, 2015 / Signed on September 24, 2015)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:
- E.coli strain WP2uvrA and Salmonella strain TA98 (absence and presence of S9-mix) and Salmonella strain TA100 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
- Salmonella strains TA1535 and TA1537 (absence and presence of S9-mix) and Salmonella strain TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of a test item film. A number of further manual counts were also required due to colonies spreading and artefacts on the plates, thus distorting the actual plate count. Occasional plates were also manually assessed for accuracy against the automated counts.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 at 5000 μg/plate in the both the presence and absence of S9-mix. Small reductions in revertant colony frequency were also noted for TA98 at 5000 μg/plate (absence of S9-mix) and for TA1537 at 5000 μg/plate (absence and presence of S9-mix). No further toxicity was noted to any of the remaining bacterial strains. Consequently, for the second mutation test the maximum recommended dose level of 5000 μg/plate was again employed as the maximum dose concentration for all of the bacterial tester strains. Results from the second mutation test (pre-incubation method) exhibited weakened bacterial background lawns in the absence of S9-mix to TA1537 from 500 μg/plate, to TA100 from 1500 μg/plate and to TA98 at 5000 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted at 5000 μg/plate to TA1537 only, although slight reductions in WP2uvrA revertant colony frequency were noted at the same maximum dose concentration. No further significant toxicity was noted to any of the remaining bacterial strains dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. An opaque test item film was noted by eye at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar, S9-mix and test item formulation used in both experiments were shown to be sterile.

See the attached document for information on tables of results

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:

- E.coli strain WP2uvrA and Salmonella strain TA98 (absence and presence of S9-mix) and Salmonella strain TA100 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA1535 and TA1537 (absence and presence of S9-mix) and Salmonella strain TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

 

Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

In Experiment 1, the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 at 5000 μg/plate in the both the presence and absence of S9-mix. Small reductions in revertant colony frequency were also noted for TA98 at 5000 μg/plate (absence of S9-mix) and for TA1537 at 5000 μg/plate (absence and presence of S9-mix). No further toxicity was noted to any of the remaining bacterial strains. In Experiment 2, weakened bacterial background lawns in the absence of S9- mix to TA1537 from 500 μg/plate, to TA100 from 1500 μg/plate and to TA98 at 5000 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted at 5000 μg/plate to TA1537 only, although slight reductions in WP2uvrA revertant colony frequency were noted at the same maximum dose concentration. No further significant toxicity was noted to any of the remaining bacterial strains dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. An opaque test item film was noted by eye at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in any of the experiments.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test (similar to OECD 474, GLP, K, rel. 2): non clastogenic, non aneugenic up to toxic concentrations

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
purity of test substance; details of acclimation; environmental conditions; evaluation criteria not reported
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
composition of test substance; details of acclimation; environmental conditions; evaluation criteria not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Samples of oleo gum resin (Commiphora molmol) was collected from a local market in Riyadh, Saudi Arabia.
Species:
mouse
Strain:
Swiss
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Care Center, King Saud University, Saudi Arabia.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 20-25 g
- Assigned to test groups randomly: Yes; animals were randomly assigned to different control and treatment groups
- Diet: Purina chow diet, ad libitum
- Water, ad libitum

ENVIRONMENTAL CONDITIONS
- Animals were maintained under standard conditions of humidity, temperature, and light (12 h light/12 h dark cycle).
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was given orally to animals as a fresh aqueous suspension.
Duration of treatment / exposure:
7 days
Frequency of treatment:
Daily
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
5 females/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Intraperitoneal
- Doses / concentrations: 100 mg/kg bw
- CP was injected 30 h before the animals were killed.
Tissues and cell types examined:
The polychromatic erythrocytes (PCE/1000 per mouse) were screened for micronuclei, and reduction of the mitotic index was assessed on the basis of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose of oleo gum resin used in the present study (500 mg/kg bw/day) had previously been reported to be pharmacologically active and was found to be effective in a preliminary study on its cytotoxic activity.

TREATMENT AND SAMPLING TIMES:
In each case, animals were killed 30 h after the last treatment. The femurs were used for a micronucleus test

DETAILS OF SLIDE PREPARATION:
The micronucleus test procedure described by Schmid was followed. The femoral cells were collected in fetal calf serum. After centrifugation, the cells were spread on slides and air-dried. Coded slides were fixed in methanol and stained in May-Gruenwald solution followed by Giemsa stain.

METHOD OF ANALYSIS:
The polychromatic erythrocytes (PCE/1000 per mouse) were screened for micronuclei, and reduction of the mitotic index was assessed on the basis of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio).
Statistics:
Statistical analysis was performed with Student's t-test.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):Test substance caused no significant difference in the incidence of micronucleated PCE in femoral cells of normal mice as compared with the controls.
- Ratio of PCE/NCE (for Micronucleus assay): There was a statistically significant decrease in the PCE/NCE ratio in the treatment groups, indicating the cytotoxic potential of test substance.
- Positive control: Cyclophosphamide significantly increased the number of micronucleated PCE and decreased the PCE/NCE ratio.

Table 7.6.2/1: Micronucleus test – results

Groups

Treatment and dose (mg/kg day)

PCE screened

Percentage of micronucleated PCE (mean ± SE)

NCE screened

PCE/NCE ratio (mean ± SE)

1

Control (Distilled water)

5996

0.43 ± 0.04

5690

1.10 ± 0.09

2

Cyclophosphamide 100

4472

4.20 ± 0.23***

6700

0.67 ± 0.10**

Test substance

3

125

5300

0.28 ± 0.09

6500

0.81 ± 0.10*

4

250

5000

0.30 ± 0.06

6600

0.76 ± 0.12*

5

500

5600

0.33 ± 0.02

8000

0.72 ± 0.08**

Groups 2, 3, 4, and 5 were statistically compared with group 1. Five mice were used in each group

*P <0.05, **P <0.01, ***P <0.001; Student's t-test

Conclusions:
Under the test conditions, test substance did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in mice.
Executive summary:

In an in vivo micronucleus test conducted similarly to OECD 474 guideline, Swiss mice (5 females/dose) were administered with test substance by oral (gavage) at the dose levels of 125, 250 and 500 mg/kg bw/day for 7 days. Vehicle control group was administered with distilled water and positive control groups were given cyclophosphamide (100 mg/kg bw, IP). Animals were sacrificed 30 h after the last treatment and the femurs were used for a micronucleus test. The polychromatic erythrocytes (PCE/1000 per mouse) were screened for micronuclei, and reduction of the mitotic index was assessed on the basis of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio).

There was a statistically significant decrease in the PCE/NCE ratio in the treatment groups, indicating the cytotoxic potential of test substance. Test substance caused no significant difference in the incidence of micronucleated PCE in femoral cells of normal mice as compared with the controls. Cyclophosphamide significantly increased the number of micronucleated PCE and decreased the PCE/NCE ratio.

 

Under the test conditions, test substance did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in mice.

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Envigo, 2016

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E. coli WP2uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Qureshi, 1993

In vivo MNT

(eq. OECD 474)

K, rel. 2

Chromosomal aberration

Mice

NA

Up to toxic concentration

Non clastogenic,

Non aneugenic

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Chromosomal aberration (Test n°2)

The clastogenic and aneugenic potential of the substance was determined using an in vivo mammalian erythrocytes micronucleus assay (Test n°2), which identifies substances that cause micronuclei in erythroblasts. These micronuclei may originate from acentric fragments or whole chromosomes, and the test thus has the potential to identify both clastogenic and aneugenic chemicals. In this study erythroblasts were sampled from femur cells of mice. None of the i.p. dose levels of up to 500 mg/kg bw, in females, induced increase in the frequency of micronucleated polychromatic erythrocytes (fMPCE), whereas the positive control chemical induced significant increases in the fMPCE. The substance was therefore considered as negative for inducing chromosomal aberrations in mice femur erythrocytes under the conditions used in this study. The substance is therefore considered as non-clastogenic and non-aneugenic.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).