Bis(glycinato)copper

Endpoint summary

Description of key information

GPMT studies:

-WoE: Basketter and Scholes, 1992, RL2, comparable to guideline study: according to Magnussen & Kligman (1970), guinea pigs. Copper chloride was not considered to be a sensitizer.

- WoE: Karlsberg et al. 1983, RL2, comparable to guideline study: guinea pig maximization test. Copper sulphate was not considered to be a sensitizer.

- Supporting study: Boman et al., 1979, RL4, abstract: according to Magnusson & Kligman, guinea pigs. Copper sulphate was regarded positive with respect to skin sensitisation

LLNA studies:

- WoE: Basketter and Scholes, 1992, RL2, comparable to guideline study: copper chloride was regarded a sensitizer.

- WoE: Ikarashi et al., 1992, RL2, well documented: Incubation with 10% CuSO₄ in 20 % ethanol resulted in a SI value of 2.67. Since the study was not conducted according to GLP and the proliferation of lymph nodes was above the set threshold of 2, the test item is considered to be a weak sensitizer.

- Glycine: QSAR estimations with VEGA and QSAR Toolbox: The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). Glycine does not meet the classification criteria. Prediction VEGA: no Sensitizer.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
The test method carried out is based on and similar to that described by Magnusson and Kligman (1970).
- Short description of test conditions: Guinea-pigs were then treated by a series of six intradermal injections in the shoulder region to induce sensitization. After 6-8 days, sensitization was boosted by a 48-hr occluded patch placed over the injection site. 12-14 days later, the animals were challenged on one flank by a 24-hr occluded patch at the maximum non-irritant concentration. Challenge sites were scored for erythema (scale 0-3) and oedema 24 and 48 hr after removal of the patches.
- Parameters analysed / observed: The classification scheme for the assessment of skin sensitization potential was based on that outlined by Magnusson and Kligman (1970).

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The results published were obtained from a GMPT test according to Magnussen & Klingman (1970). Although GLP compliance was not reported the method is considered to be reliable and the results are expected to provide useful information about the sensitizing potential of copper bisglycinate in a Weight-of-Evidence approach. Thus, for this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

not specified

Details on test animals and environmental conditions:

animals weighed approximately 350 g, other details on test animals and environmental conditions were not reported

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

0.0001%

Day(s)/duration:

6-8

Adequacy of induction:

other: six intradermal injections in the shoulder region to induce sensitisation

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

1.0%

Day(s)/duration:

2

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

0.25%

Day(s)/duration:

after 12-14 days

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

not reported

Details on study design:

not reported

Challenge controls:

not reported

Positive control results:

not reported

Key result

Reading:

other: reading time points not reported

Group:

test chemical

Dose level:

0.0001%

No. with + reactions:

0

Remarks on result:

other: only the final result was reported

Remarks:

No time points for reading nor exact values were reported

Key result

Reading:

other: No time points for reading were reported

Group:

negative control

Remarks on result:

other: results of negative control were not reported

Key result

Reading:

other: No time points for reading were reported

Group:

positive control

Remarks on result:

other: No results of a positive control were reported

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present test conducted according to Magnussen & Kligman (1970), guinea pigs were treated intradermally and epicutaneously with copper chloride at concentrations of 0.0001% (i.c.), 0.1% (e.c.) and challenged with 0.25% copper chloride. None of the treated animals showed a response to copper chloride, thus copper chloride was not considered to be a sensitizer.

Executive summary:

In the publication by Basketter and Scholes, 1992, a test was conducted according to Magnussen & Kligman (1970). Guinea pigs were treated intradermally and epicutaneously with copper chloride at concentrations of 0.0001% (i.c.), 0.1% (e.c.) and challenged with 0.25% copper chloride. The guinea-pigs were treated with a series of six intradermal injections in the shoulder region to induce sensitization. After 6-8 days, sensitization was boosted by a 48-hr occluded patch placed over the injection site. 12-14 days later, the animals were challenged on one flank by a 24-hr occluded patch at the maximum non-irritant concentration. Challenge sites were scored for erythema (scale 0-3) and oedema 24 and 48 hr after removal of the patches. The classification scheme for the assessment of skin sensitization potential was based on that outlined by Magnusson and Kligman (1970).

None of the treated animals showed a response to copper chloride, thus copper chloride was not considered to be a sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A guinea pig-maximization test (GMPT) was conducted.
- Short description of test conditions: 3 studies with varying concentrations of copper sulphate at intradermal induction (0.1%, 0.05% and 0.01%), were carried out. At epicutaneous induction 25 % of copper sulphate salt in petrolatum was used in all series. Challenge testing was performed on day 21 with 1.0, 0.5 and 0.1% of the salt in pet. The animals exposed to 0.1% copper sulphate at intradermal induction were also challenged with nickel sulphate at 0.5, 0.25 and 0.05% in pet.
- Parameters analysed / observed: Parameters as described for the GMPT test were analsysed.

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A GPMT is available, which is considered reliable with restrictions as an adequate supporting information for classification and labelling purposes. Futhermore several other studies are available which are considered in a Weight of Evidence approach. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

Further details on test animals and environmental conditions were not reported

Route:

intradermal

Vehicle:

petrolatum

Concentration / amount:

0.1, 0.05, 0.01%

Adequacy of induction:

not specified

Route:

other: epicutaneous

Vehicle:

petrolatum

Concentration / amount:

25%

Day(s)/duration:

20

Adequacy of induction:

not specified

No.:

#1

Route:

other: not specified

Vehicle:

petrolatum

Concentration / amount:

0.1% copper sulphate

Adequacy of challenge:

not specified

No.:

#2

Route:

other: not specified

Vehicle:

petrolatum

Concentration / amount:

0.5, 0.25, 0.05% nickel sulphate

Adequacy of challenge:

not specified

No. of animals per dose:

20

Details on study design:

Details on study design were not reported

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1% i.c., 25% e.c.

Remarks on result:

other: treatment not significant different from control

Remarks:

no exact values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1% i.c., 25% e.c.

Remarks on result:

other: treatment not significant different from control

Remarks:

no values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.1% i.c., 25% e.c.; Challenge 1, 0.5, 0.1%

Remarks on result:

other: No values were reported

Remarks:

The maximum number of reactions was one animal per challenge concentration. For further information please refer to the 'any other information on results incl. tables' section.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1% i.c., 25% e.c.; Challenge 1, 0.5, 0.1 %

Remarks on result:

other: No values were reported

Remarks:

No positive control results were reported

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.05% i.c. 25% e.c.

Remarks on result:

other: treatment not significant different from control

Remarks:

no values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.05% i.c., 25% e.c.

Remarks on result:

other: treatment not significant different from control

Remarks:

no values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.05% i.c., 25% e.c.; Challenge 1, 0.5, 0.1%

Remarks on result:

other: No values were reported

Remarks:

The maximum number of reactions was one animal per challenge concentration. For further information please refer to the 'any other information on results incl. tables' section.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.05% i.c., 25% e.c., Challenge 1, 0.5, 0.1%

Remarks on result:

other: No values were reported

Remarks:

No positive control results were reported

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.01& i.c., 25% e.c.

Remarks on result:

other: treatment not significant different from control (challenge with nickel sulphate)

Remarks:

no values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.01% i.c., 25% e.c.

Remarks on result:

other: treatment not significant different from control (challenge with nickel sulphate)

Remarks:

no values have been reported, instead any significant difference was reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.01% i.c., 25% e.c.; Challenge 1, 0.5, 0.1%

Remarks on result:

other: No values were reported

Remarks:

The maximum number of reactions was one animal per challenge concentration. For further information please refer to the 'any other information on results incl. tables' section.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.01% i.c., 25% e.c., Challenge 1, 0.5, 0.1%

Remarks on result:

other: No valures were reported

Remarks:

No positive control results were reported

Sensitization and testing of guinea pigs according to GPMT:

Induction	CuSO4 x 5 H2O	time (h)
i.c.	0.1%
e.c.	25%
Challenge concentration (%w/w)			1	0.5	0.1	Control (pet.)
Controls		24	0	1	1	1
n=18		48	1	1	0	1
Exposed n=19		24 & 48	0	0	0	0
		24	-	N.S.	N.S.	N.S.
		48	N.S.	N.S.	-	N.S.

- not analyzed; N.S. not significant.

Induction	CuSO4 x 5 H2O	time (h)
i.c.	0.05%
e.c.	25%
Challenge concentration (%w/w)			1	0.5	0.1	Control (pet.)
Controls		24	0	0	0	0
n=20		48	1	1	2	1
Exposed n=19		24	0	0	0	0
		48	1	0	0	0
		24	-	-	-	-
		48	-	N.S.	N.S.	N.S.

- not analyzed; N.S. not significant.

Induction	CuSO4 x 5 H2O	time (h)
i.c.	0.01%
e.c.	25%
Challenge concentration (%w/w)			1	0.5	0.1	Control (pet.)
Controls		24 & 48	0	0	0	0
n=20
Exposed n=19		24 & 48	0	0	0	0

Interpretation of results:

study cannot be used for classification

Conclusions:

In a guinea pig maximization test animals were intradermally treated with 0.1%, 0.05% and 0.01% copper sulphate and epicutaneously with 25% copper sulphate. The animals were challenged at day 21 after treatment with either nickel sulphate (0.5, 0.25, 0.05%) or copper sulphate (1.0, 0.5, 0.1%). All treatments were conducted in petrolatum. No significant differences were detected between the control animals and the treated animals with regard to the parameters analysed in the guinea pig maximization test. Based on the presented results copper sulphate is not considered to be a sensitizer.

Executive summary:

In the publication by Karlberg et al., 1983, a guinea pig-maximization test (GMPT) was conducted. Studies with varying concentrations of copper sulphate at intradermal induction (0.1%, 0.05% and 0.01%), were carried out. 20 animals per dose were used. At epicutaneous induction 25 % of copper sulphate salt in petrolatum was used in all series. Challenge testing was performed on day 21 with 1.0, 0.5 and 0.1% of the salt in petrolatum. The animals exposed to 0.1% copper sulphate at intradermal induction were also challenged with nickel sulphate at  0.5, 0.25 and 0.05% in petrolatum. Parameters as described for the GMPT test were analsysed.

No significant differences were detected between the control animals and the treated animals with regard to the parameters analysed in the guinea pig maximization test. Based on the presented results, copper sulphate is not considered to be a sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Test according to the principle of Magnusson & Kligman 1970.
- Short description of test conditions: Two groups of 20 animals were used for each metal compound. One group was actively sensitiactl and the other group (control was treated in the same way as the experimental group (Freund's complete adjuvant, petrolatum occlusion. etc.) except for the test compound. The groups were kept separately in plastic cages. challenged simultaneously and the readings performed blind.
- Parameters analysed / observed: Number of positive animals (as scored according to Magnusson & Kligman (1970)) for each concentration is given.

GLP compliance:

no

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

Animals were kept in plastic cages, size was not reported. No further details on test animals and environmental conditions were given.

Route:

intradermal

Vehicle:

petrolatum

Concentration / amount:

0.01%

Adequacy of induction:

other: no information on concentration finding test, duration was also not reported

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25%

Adequacy of induction:

other: no information on concentration finding tests, duration was not reported

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

1.0, 0.5, 0.1%

Adequacy of challenge:

other: no information on concentration finding tests, duration was not reported

No. of animals per dose:

10

Details on study design:

No further details on study design reported

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1% challenge concentration

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1% challenge concentration

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

study cannot be used for classification

Conclusions:

The test described in the abstract of Boman et al., 1979, was conducted according to Magnusson & Kligman (1970). Groups of 10 guinea pigs were treated intradermally and epicutaneously with 0.01% and 25%, respectively. 21 days after the intradermal application the animals were challenged with 1.0, 0.5, and 0.1% copper sulphate solution in petrolatum. After 24 h (first reading 2 animals were scored positive for sensitisation and after 48h 7 animals were scored positive for skin sensitisation, thus, copper sulphate was regarded positive with respect to skin sensitisation.

Executive summary:

The test described in the abstract of Boman et al., 1979, was conducted according to Magnusson & Kligman (1970). Groups of 10 guinea pigs were treated intradermally and epicutaneously with 0.01% and 25%, respectively. 21 days after the intradermal application the animals were challenged with 1.0, 0.5, and 0.1% copper sulphate solution in petrolatum. After 24 h (first reading) 2 animals were scored positive for sensitisation and after 48h 7 animals were scored positive for skin sensitisation, thus, copper sulphate was regarded positive with respect to skin sensitisation.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
The assay was carried out as described elsewhere (Basketter et al., 1991).
- Short description of test conditions:
CBA/Ca mice were used at the age of 8-12 wk. Animals of both sexes were used, but single experiments were limited to one sex. Groups of four mice were treated by a daily topical application of 25 µL of each concentration on the dorsal surface of each ear for 3 consecutive days. Control mice were treated with the vehicle alone. 4-5 days after the first topical application, all mice were injected intravenously through the tail vein with 250 µL phosphate buffered saline (PBS) containing [3H]methyl thymidine (3HTdR; 20 µCi). After 5 hr the mice were killed by carbon dioxide asphyxiation, and the draining auricular lymph nodes were excised and pooled for each experimental group. A single-cell suspension of lymph node cells (LNC) was prepared. After an overnight incubation with TCA at 4°C, the precipitate was recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10ml scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting.
- Parameters analysed / observed: test to control lymphocyte proliferation ratio

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks

Vehicle:

dimethyl sulphoxide

Concentration:

Concentrations were from the following range: 100, 50, 25, 10, 5, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05 and 0.01%. Used were: 1, 2.5, 5%

No. of animals per dose:

4

Details on study design:

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION: Groups of four mice were treated by a daily topical application of 25 µL of each concentration on the dorsal surface of each ear for 3 consecutive days. Control mice were treated with the vehicle alone. 4-5 days after the first topical application, all mice were injected intravenously through the tail vein with 250 µL phosphate buffered saline (PBS) containing [3H]methyl thymidine (3HTdR; 20 µCi). After 5 hr the mice were killed by carbon dioxide asphyxiation, and the draining auricular lymph nodes were excised and pooled for each experimental group. A single-cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless-steel gauze (200-mesh size), using the plunger of a syringe. Pooled LNC were pelleted at 190g for 10 min, washed twice with 10 ml PBS and resuspended in 3 ml trichloroacetic acid (TCA; 5%) for the precipitation of macromolecules. After an overnight incubation with TCA at 4°C, the precipitate was recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10ml scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per min per lymph node (dpm/node), and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

Key result

Parameter:

SI

Remarks:

SI value was not determined, instead ratio of test to control lymphocyte proliferation was calculated

Value:

8.1

Test group / Remarks:

1%

Remarks on result:

other:

Remarks:

positive indication of sensitisation

Key result

Parameter:

SI

Remarks:

SI value was not determined, instead ratio of test to control lymphocyte proliferation was calculated

Value:

13.8

Test group / Remarks:

2.5%

Remarks on result:

other:

Remarks:

positive indication of sensitisation

Key result

Parameter:

SI

Remarks:

SI value was not determined, instead ratio of test to control lymphocyte proliferation was calculated

Value:

13.6

Test group / Remarks:

5%

Remarks on result:

other:

Remarks:

positive indication of sensitisation

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present test conducted as described in Basketter and Scholes (1992), copper chloride was regarded a sensitizer in the LLNA.

Executive summary:

In the present test conducted as described in Basketter and Scholes (1992), mice were exposed to 1%, 2.5% and 5 % copper chloride in DMSO applied topically on the dorsal surface of each ear for 3 consecutive days. Control mice were treated with the vehicle alone. 4-5 days after the first topical application, all mice were injected intravenously through the tail vein with 250 µL phosphate buffered saline (PBS) containing [³H]methyl thymidine (³HTdR; 20 µCi). After 5 h the mice were killed and a single-cell suspension of lymph node cells (LNC) was prepared. ³HTdR incorporation was measured by B-scintillation counting. The T/C ratio of lymph nodes was determined and copper chloride was found to be sensitizing for each concentration tested with the following values: 1% = 8.1; 2.5% = 13.8; 5% = 13.6.

Thus, under the conditions of the test copper chloride was regarded a sensitizer.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: induction of LNC proliferation in LLNA, similar to OECD guideline 429
- Short description of test conditions: mice (n I 3) received 25 µL of test chemical solution, or vehicle alone, on the dorsum of each ear. The application was repeated for three consecutive days. In some experiments, the dorsal surface of each ear was gently abraded by lightly dragging a 19-g needle across the dorsal surface of each ear five times (without causing bleeding) just prior to the application of test chemicals. Four days following the initial application, mice were killed and the draining lymph nodes (auricular and axillary) were excised and pooled per animal. After incubation with [3H]TdR for 18h the cultured cells were harvested and the incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) i standard deviation per node of three animals for each test group. Increases in [3H]TdR incorporation relative to vehicle-treated controls were calculated for each test group, and expressed as stimulation indices (SI).

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6-8 weeks

Vehicle:

other: 20% ethanol solution

Concentration:

10% CuSO4

No. of animals per dose:

3

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The mice (n = 3) received 25 µL of test chemical solution, or vehicle alone, on the dorsum of each ear. The application was repeated for three consecutive days. In some experiments, the dorsal surface of each ear was gently abraded by lightly dragging a 19-g needle across the dorsal surface of each ear five times (without causing bleeding) just prior to the application of test chemicals. Four days following the initial application, mice were killed and the draining lymph nodes (auricular and axillary) were excised and pooled per animal. A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200-mesh steel gauge. Lymphocyte suspensions were washed twice in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10% fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), 100 µg/ml penicillin
and 100 U/ml streptomycin. The cell concentration was adjusted to give 5 E+06 cells/mL.. Lymphocyte suspensions were seeded into 96-well microtiter plates at a concentration of 1 E+06 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H]methyl thymidine ([3H]TdR) for 18 h at 37°C in a humidified atmosphere of 5% CO2 in air.
Culture was terminated by automatic cell harvesting. The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group. Increases in [3H]TdR incorporation relative to vehicle-treated controls were calculated for each test group, and expressed as stimulation indices (SI).

Key result

Parameter:

SI

Value:

2.67

Test group / Remarks:

10% CuSO4/ lymphocyte proliferation in 3 treated mice compared to 3 control mice

Parameter:

SI

Test group / Remarks:

negative control

Remarks on result:

other: not reported

Parameter:

SI

Test group / Remarks:

positive control

Remarks on result:

other: not reported

Cellular proliferation data / Observations:

In the present study only the results from one treatment group were reported.

THE LOCAL LYMPH NODE ASSAY PERFORMED WITH FIVE METAL SALTS

Chemical	[3H]TdR incorporation (mean cpm±SD (x 10^-3)	SI
20% EtOH	1.52± 0.67	-
10% FeSO4	2.03±0.63	1.32
10% MnCl2	1.26±0.08	0.82
10% ZnSO4	2.14±0.77	1.41
10% CuSO4	4.08±1.20	2.67
10% NiSO4	3.56±0.25	2.32

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present study conducted by Ikarashi et al. (1992), copper sulfate is considered to be a weak sensitizer in the LLNA.

Executive summary:

In the present study conducted by Ikarashi et al. (1992), copper sulfate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [³H]TdR. After the incubation period the [³H]TdR incorporation was detected and the SI was determined.

Incubation with 10% CuSO₄ in 20% ethanol resulted in a SI value of 2.67. Thus, under the conditions of the present test the substance was considered a sensitizer, however, according to OECD guideline 429, a SI value < 3 does not result in a classification as sensitizer.

Since the study was not conducted according to GLP and the proliferation of lymph nodes was above the set threshold of 2, copper sulfate is considered to be a weak sensitizer.

Reference 6

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: Information from the QSAR Toolbox database

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

not specified

Vehicle:

not specified

Key result

Parameter:

EC3

Remarks on result:

other: Value not reported in QSAR Toolbox

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

mean of all tested groups

Remarks on result:

other:

Remarks:

Result as reported by the OECD QSAR Toolbox

Interpretation of results:

GHS criteria not met

Conclusions:

The OECD QSAR Toolbox was used in order to gather all available information about the skin sensitizing potential of glycine. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). Glycine does not meet the classification criteria of Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) and is therefore not classified as dermal sensitizer.

Executive summary:

No data are available regarding the skin sensitizing potential of glycine. Beside the QSAR prediction of skin sensitization using the VEGA-Module data was also gathered from the OECD QSAR Toolbox. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). In both cases glycine was niot classified as skin sensitizer because there was no SI value above 3 in the mentioned study reports, thus, glycine is not considered to be a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In the publication by Basketter and Scholes, 1992, a test was conducted according to Magnussen & Kligman (1970). Guinea pigs were treated intradermally and epicutaneously with copper chloride at concentrations of 0.0001% (i.c.), 0.1% (e.c.) and challenged with 0.25% copper chloride. The guinea-pigs were treated with a series of six intradermal injections in the shoulder region to induce sensitization. After 6-8 days, sensitization was boosted by a 48-hr occluded patch placed over the injection site. 12-14 days later, the animals were challenged on one flank by a 24-hr occluded patch at the maximum non-irritant concentration. Challenge sites were scored for erythema (scale 0-3) and oedema 24 and 48 hr after removal of the patches. The classification scheme for the assessment of skin sensitization potential was based on that outlined by Magnusson and Kligman (1970).

None of the treated animals showed a response to copper chloride, thus copper chloride was not considered to be a sensitizer.

In the publication by Karlberg et al., 1983, a guinea pig-maximization test (GMPT) was conducted. Studies with varying concentrations of copper sulphate at intradermal induction (0.1%, 0.05% and 0.01%), were carried out. 20 animals per dose were used. At epicutaneous induction 25 % of copper sulphate salt in petrolatum was used in all series. Challenge testing was performed on day 21 with 1.0, 0.5 and 0.1% of the salt in petrolatum. The animals exposed to 0.1% copper sulphate at intradermal induction were also challenged with nickel sulphate at  0.5, 0.25 and 0.05% in petrolatum. Parameters as described for the GMPT test were analsysed.

No significant differences were detected between the control animals and the treated animals with regard to the parameters analysed in the guinea pig maximization test. Based on the presented results, copper sulphate is not considered to be a sensitizer.

The test described in the abstract of Boman et al., 1979, was conducted according to Magnusson & Kligman (1970). Groups of 10 guinea pigs were treated intradermally and epicutaneously with 0.01% and 25%, respectively. 21 days after the intradermal application the animals were challenged with 1.0, 0.5, and 0.1% copper sulphate solution in petrolatum. After 24 h (first reading) 2 animals were scored positive for sensitisation and after 48h 7 animals were scored positive for skin sensitisation, thus, copper sulphate was regarded positive with respect to skin sensitisation.

In the present test conducted as described in Basketter and Scholes (1992), mice were exposed to 1%, 2.5% and 5 % copper chloride in DMSO applied topically on the dorsal surface of each ear for 3 consecutive days. Control mice were treated with the vehicle alone. 4-5 days after the first topical application, all mice were injected intravenously through the tail vein with 250 µL phosphate buffered saline (PBS) containing [³H]methyl thymidine (³HTdR; 20 µCi). After 5 h the mice were killed and a single-cell suspension of lymph node cells (LNC) was prepared. ³HTdR incorporation was measured by B-scintillation counting. The T/C ratio of lymph nodes was determined and copper chloride was found to be sensitizing for each concentration tested with the following values: 1% = 8.1; 2.5% = 13.8; 5% = 13.6.

Thus, under the conditions of the test copper chloride was regarded a sensitizer.

In the study conducted by Ikarashi et al. (1992), copper sulfate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [³H]TdR. After the incubation period the [³H]TdR incorporation was detected and the SI was determined. Incubation with 10% CuSO₄ in 20% ethanol resulted in a SI value of 2.67. Thus, under the conditions of the present test the substance was considered a sensitizer, however, according to OECD guideline 429, a SI value < 3 does not result in a classification as sensitizer. Since the study was not conducted according to GLP and the proliferation of lymph nodes was above the set threshold of 2, copper sulfate is considered to be a weak sensitizer.

No data are available regarding the skin sensitizing potential of glycine. Beside the QSAR prediction of skin sensitization using the VEGA-Module data was also gathered from the OECD QSAR Toolbox. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). In both cases glycine was not classified as skin sensitizer because there was no SI value above 3 in the mentioned study reports, thus, glycine is not considered to be a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the GPMTs performed by Basketter and Scholes (1992), Karlsberg et al. (1983) and Boman et al. (1979) it is concluded that copper is a weak sensitizer. At concentrations of 1% CuSO₄ indications for sensitisation were observable. At lower concentrations of metal salts (i.e. 0.1, 0.05 and 0.01% CuSO₄ and 0.0001% CuCl₂) no sensitising potential for copper ions was observed. It can be assumed that a higher concentration of copper is taken up by an organism via bis(glycinato)copper. It is known that copper may induce allergic reactions, whereas the other ingredient of the target substance bis(glycinato)copper, glycine, is expected not to induce allergic reactions.

Based on the data from GPMT studies, bisglycinatocopper is classified as weak sensitizer for precautionary reasons.

The skin sensitisation potential of copper salts was investigated in two studies using either CuCl₂ (1, 2.5 and 5% in DMSO; Basketter et al., 1992) or CuSO₄ (10% in 20% ethanol; Ikarashi et al., 1992) for topical application. Four to five days after the first application the draining lymph nodes were excised and proliferation was measured with [³H]TdR incorporation. Basketter et al. detected an increase of proliferation and determined the test to control proliferation ratio. The increase of the ratio was correlated to the increasing concentrations (1% CuCl₂= 8.1; 2.5% CuCl₂= 13.8; 5% CuCl₂= 13.6). Ikarashi et al. reported a SI value of 2.67 and based on this increase in proliferation considered that CuSO₄ is a sensitizer. However, assuming the conduction of the test was similar to that described in OECD guideline 429 it should be kept in mind that the threshold for determination of a sensitizer recommended by OECD guideline 429 is a SI value of 3, 10% CuSO₄ would not have been classified as sensitizer according to OECD guideline. Nevertheless, based on the other information available and for precautionary reasons copper is considered to be a weak sensitizer.

Taken together, the target substance bisglycinatocopper is also considered to be a weak sensitizer because the copper ion is expected to be the species that determines the sensitisation potential.

For precautionary reasons, bis(glycinato)copper is classified as Category 1B (indication of skin sensitising potential) according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).