Reaction mass of 3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate and 3,7-dimethyloctyl 3-methyl-2-butenoate and citronellyl 3-methylcrotonate

Endpoint summary

Description of key information

Not skin irritant (OECD 406 skin irritation pre-test)

Not eye irritant (OECD 437, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 9, 1983 to April 2, 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 406 skin irritation pre-test

Principles of method if other than guideline:

As part of a Magnusson-Kligman guinea pig maximization, shaved clilpped shoulders of male guinea pigs were patched with 25% dilution of the test sbstance in vaseline for 48 hours under occlusion. Sinodor induced slight skin irritation in 5/10 guinea pigs.

In a primary irritation study, Pirbright white guinea pigs weighing 300-500 g were topically treated on the shorn flank daily with 50 % test material olive oil for 5 days. The test solution was rubbed in for 30 seconds at each application. Animals were individually housed and provided feed and water ad libitum. The room temperature was 20 °C with a humidity of 50 % and a dark/light cycle of 12 hours.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

A 200ml sample of the test subtsance was received from the sponsor on December 17, 1982. It was a clear colourless liquid, designated: Sinodor X-09648, No. 211234, date 13.12.82.

Species:

guinea pig

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: Male
- Weight at study initiation: 200 - 400 g
Main test is conducted with 15 young male SPF bred albino guinea pigs (body weight 200-400g) obtained from the central intitute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands.
The experiment is preceded by an acclimatization period of at least two days to accustom the animals to the environmen-tal conditions prevailing in our laboratory.
The guinea pigs are kept under conventional conditions and individually housed in suspended stainless steel cages, fitted with wire mesh floors and fronts. The temperature in the animal room is kept at 21 +/- 1 °C, the relative humidity at 40% at least and a 12 h light/dark cycle is maintained.
The guinea pigs are fed pelleted stock diet from Hope Farms (Woerden, The Netherlands). The diet as well as tap water are provided ad libitum throughout the experiment.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

other: vaseline

Controls:

yes

Amount / concentration applied:

FOR TEST GROUP:
1. Induction phase:
- Intradermal applications: 10% Sinodor in propylene glycol, 10% in mixture of FCA and propylene glycol (1:1), FCA only
- Topical application: 25% in vaseline

Challenge: 10% in vaseline

FOR CONTROL GROUP:
1. Induction phase:
- Intradermal applications: FCA only, propylene glycol only, FCA+propylene glycol (1:1) only
- Topical application: vaseline only

2. Challenge: 10% in vaseline

Duration of treatment / exposure:

The diluted test solution in propylene glycol and FCA+propylene glycol was applied during 24 hours.
The diluted test solution in vaseline was applied during 48 hours.
The diluted test solution in vaseline during challenge was applied during 24 hours

Observation period:

Maximum 48 hours

Number of animals:

Test group: 10 animals
Control group: 5 animals

Details on study design:

The test was divided into two stages:
a) induction treatment by intradermal injection and topical application
b) challenge treatment by topical application

Induction is effected in a two-stage operation consisting of, firstly, three pairs of intradermal injections made simultane-ously and, secondly, one week later, a closed patch exposure performed over the injection sites. For this purpose an area of c. 24 cm2 of dorsal skin in the shoulder region is clipped free of hair with electric clippers.

The induction by intradermal injection was performed in two rows of each three injection sites in the shoulder region as follows:
Test animals:
- two injections (0.1 ml) of Freund's Complete Adjuvant (FCA)
- two injections (0.1 ml) of a 10% dilution (w/v) of Sinodor in propylene glycol (PG)
- two injections (0.1 ml) of a 10% dilution (w/v) of Sinodor in FCA and PG (1:1)
Control animals:
- two injections (0.1 ml) of Freund's Complete Adjuvant (FCA)
- two injections (0.1 ml) of propylene glycol (PG)
- two injections (0.1 ml) of FCA and PG (1:1)

Skin readings are made 24h after the treatment.

One week after the intradermal injections, the induction by topical application was made in the same shoulder region. The test animals were treated with a 25% dilution (w/w) of Sinodor in vaseline. The controls were similarly treated with vaseline alone.
The test animals are treated as follows: a 2x4cm patch of Whatman No 3 MM filter paper is loaded with the appropriate mixture of the test substance and carrier. The patch is placed over the sites of the intradermal injections and covered with a piece of PVC foil and a piece of Leukopor hypo-allergic paper bandage. This, in turn, is secured with a 7.5cm wide Tensoplast bandage. The dressing is left in place for 48h. The control animals are similarly treated with patches with car-rier only. Skin readings are made after removal of the patches.

The challenge was carried out two weeks after the topical induction in both test and control animals. The right flank of all animals was topically treated with a 10% dilution (w/w) of Sinodor in vaseline.
An area of 5x5 cm on the left flank of each animal is clipped free of hair and closely shaved. Subsequently a Silverpatch is loaded with the appropriate mixture of the test substance and carrier and place in the centre of the shaved area. The patch is covered with Leukopor bandage which is held in place by Tensoplast. The control animals are also treated with the test substance. The patches are left in place for 24 h. Skin readings are made immediately after removal of the patches and 24 and 48h thereafter.

Irritation parameter:

erythema score

Time point:

48 h

Remarks on result:

probability of weak irritation

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Remarks:

As part of a Magnusson-Kligman guinea pig maximization, shaved clilpped shoulders of male guinea pigs were patched with 25% citronellyl-3-methyl-2-butenoate in vaseline for 48 hours under occlusion. Citronellyl-3-methyl-2-butenoate induced slight skin irritation in 5/10 guinea pigs.

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Remarks:

As part of a Magnusson-Kligman guinea pig maximization, shaved clilpped shoulders of male guinea pigs were patched with 25% citronellyl-3-methyl-2-butenoate in vaseline for 48 hours under occlusion. Citronellyl-3-methyl-2-butenoate induced slight skin irritation in 5/10 guinea pigs.

Irritant / corrosive response data:

The topical applications with the 25% dilution of the test substance in vaseline, made in the induction phase, induced erythema in five out of ten animals.
Vaseline alone did not induce skin reactions in any of the control animals.

Interpretation of results:

not irritating

Conclusions:

As part of a Magnusson-Kligman guinea pig maximization, shaved clilpped shoulders of male guinea pigs were patched with 25% citronellyl-3-methyl-2-butenoate in vaseline for 48 hours under occlusion.
Citronellyl-3-methyl-2-butenoate induced slight skin irritation in 5/10 guinea pigs.
Not sufficient for classification.

Executive summary:

The test substance Sinodor was examined for possible sensitization potential by a maximization test in guinea pigs.

From the reaction to the challenge treatment with a 10% dilution of the test substance in vaseline, it was concluded that the test substance did not induce delayed contact hypersensitivity in guinea pigs under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 07, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: SINODOR CQ U/A - 6099932
Batch-No.: VE00172025
Purity: > 97% (sum isomers)
Stability in Solvent: Not relevant
Storage: At room temperature, protected from light and moisture
Expiration Date: May 2013

Species:

other: Bovine eyes

Strain:

other: Corneas

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the
abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were
transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at
ambient temperature. The corneae were isolated on the same day after delivery of the eyes
and used directly for the BCOP test.
All eyes were carefully examined macroscopically for defects. Those presenting defects such
as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of
tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in
the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and
checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the
description given in OECD guideline 437, annex III, that consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea,
respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but
stretching was avoided. After the anterior part of the holder was positioned on top of the
cornea and fixed in place with screws, both compartments of the holder were filled with
complete medium. The posterior compartment was filled first to return the cornea to its
natural convex position. Care was taken to assure no air bubbles were present within the
compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour
at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

The anterior compartment received the test item or negative or positive control at a volume
of 0.75 mL on the surface of the corneae.
The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat.
Saline was used as negative control item.

Duration of treatment / exposure:

10 minutes
120 minutes

Number of animals or in vitro replicates:

Negative control: 3 Corneae
Positive control: 3 Corneae
Test item: 3 Corneae

Details on study design:

The experiment was performed to determine an irritation effect of the test item on the
corneal opacity.
The anterior compartment received the test item or negative or positive control at a volume
of 0.75 mL on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath,
while the corneae were in a horizontal position.
The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat.
Saline was used as negative control item.
The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side
with saline, fresh cMEM was added into the anterior compartment. The corneae were then
incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a 2nd opacity
reading (t130).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Nafluorescein
solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was
placed in the anterior compartment. Corneae were incubated again in a horizontal position
for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot
of the mixed complete medium from the posterior chamber was measured
spectrophotometrically at 490 nm (OD490).

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae,
and displays a numerical opacity value. This value was recorded in a table. The
opacitometer was calibrated as described in the manual and the opacity of each of the
corneae was determined by reading each holder placed in the photoreceptor compartment
for treated cornea.
The basal opacity of all corneae was recorded. Each corneae with a value of the basal
opacity > 7 was discarded. Sets of three corneae were used for treatment with the test items
and the negative and positive controls.
Complete medium was completely removed from the anterior compartment and replaced by
the test item, positive or negative control. The anterior compartment was plugged. The
cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of
the cornea with the test item and was incubated in a water-bath at 32 ± 1 °C for ten minutes
followed by two hours incubation after replacing the test item solution by cMEM in a vertical
position. Afterwards, the opacity value was determined again.

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of
the integrity of the epithelial cell sheets. After the final opacity measurement was performed,
the complete medium was removed from the anterior compartment and replaced by 1 mL of
a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a
horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the
posterior compartment was removed, well mixed and the optical density at 490 nm (OD490)
was determined with a spectrophotometer.

Criteria for Determination of a Valid Test
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the
in vitro irritation score of the negative control was ≤ 3.

Irritation parameter:

in vitro irritation score

Value:

3.01

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corneal Opacity :
The opacity reading for the test article was 2.67, 1.67 and 4.67.
The mean opacity reading for the negative control was 1.33.
The opacity reading for the positive control was 78.67, 74.67 and 81.67.

Corneal Permeability:
The optical density for the test article was 0.004, 0.001 and -0.003.
The mean optical density for the negative control was 0.048.
The optical density for the positive control was 0.486, 0.209 and 0.318.

Interpretation of results:

not classified

Conclusions:

With the negative control (saline) neither an increase of opacity nor permeability of the
corneae could be observed (mean in vitro irritation score 2.05).
The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of
the corneae (mean in vitro irritation score 83.40) corresponding to a classification as
corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item SINODOR CQ U/A - 6099932 caused a very
slight increase of the corneal opacity, whereas a rise of the permeability values was not
observed. The calculated mean in vitro irritation score was 3.01 (threshold for corrosivity /
severe irritancy: ≥ 55.1). According to OECD 437 the test item is classified as not corrosive /
not severe irritant to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of

SINODOR CQ U/A - 6099932 by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item

SINODOR CQ U/A - 6099932, the positive, and the negative controls were applied to

corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM

medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum

(FCS) (complete medium = cMEM). After the incubation phase the test item, the positive,

and the negative controls were each rinsed from the corneae. Further, the corneae were

incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was

measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring

spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal

position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase

of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of

the corneae corresponding to a classification as corrosive / severe irritant to the eye

(CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item SINODOR CQ U/A - 6099932 caused a very

slight increase of the corneal opacity. Permeability effects were not observed. The calculated

mean in vitro irritation score was 3.01. According to OECD 437 the test item is classified as

not corrosive / not severe irritant to the eye.

In conclusion, according to the current study and under the experimental conditions

reported, the test item SINODOR CQ U/A - 6099932 is not corrosive / not severe irritant to

the eye (CLP/EPA/GHS (Cat 1)).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Since none of the test animals showed erythema reactions in the challenge test at the 24 hours reading, it is concluded that the test substance did not induce delayed contact hypersensitivity in any of the guinea pigs under the conditions of the test.

Eye irritation (OECD 437, GLP):

This in vitro study was performed to assess the corneal irritation and damage potential of SINODOR CQ U/A - 6099932 by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item SINODOR CQ U/A - 6099932, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item SINODOR CQ U/A - 6099932 caused a very slight increase of the corneal opacity. Permeability effects were not observed. The calculated mean in vitro irritation score was 3.01. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

In conclusion, according to the current study and under the experimental conditions reported, the test item SINODOR CQ U/A - 6099932 is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown no skin irritancy potential and should therefore not be classified according to the (EC) No 1272/2008 Regulation (CLP).

Based on the data available and key results described in this summary, the substance has shown no eye irritancy potential and should therefore not be classified according to the (EC) No 1272/2008 Regulation (CLP).