Registration Dossier

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.66 mg/m³

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

Overall assessment factor (AF):

75

Dose descriptor starting point:

NOAEL

Value:

20 mg/kg bw/day

Modified dose descriptor starting point:

NOAEC

Value:

49.4 mg/m³

AF for dose response relationship:

1

Justification:

default value (ECHA)

AF for differences in duration of exposure:

6

Justification:

default value (ECHA)

AF for interspecies differences (allometric scaling):

1

Justification:

default value (ECHA)

AF for other interspecies differences:

2.5

Justification:

default value (ECHA)

AF for intraspecies differences:

5

Justification:

default value (ECHA)

AF for the quality of the whole database:

1

Justification:

default value (ECHA)

AF for remaining uncertainties:

1

Justification:

see 'Discussion'

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

3.3 mg/m³

DNEL extrapolated from long term DNEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.093 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

Overall assessment factor (AF):

72

Dose descriptor starting point:

NOAEL

Value:

20 mg/kg bw/day

AF for dose response relationship:

1

Justification:

see 'Discussion'

AF for differences in duration of exposure:

6

Justification:

default value (ECHA)

AF for interspecies differences (allometric scaling):

4

Justification:

default value (ECHA)

AF for other interspecies differences:

2.5

Justification:

default value (ECHA)

AF for intraspecies differences:

5

Justification:

default value (ECHA)

AF for the quality of the whole database:

1

Justification:

see 'Discussion'

AF for remaining uncertainties:

1

Justification:

see 'Discussion'

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.46 mg/kg bw/day

DNEL extrapolated from long term DNEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

2-Mercaptomethylbenzimidazole MB2 (CAS 53988-10-6)

and its Na+ salt (CAS 75045-07-7)

DNELs (worker)

Repeated dose toxicity

A subacute oral gavage study rats was evaluated for the derivation of DNELs of MB2 (2-mercapto-methylbenzimidazole; also named MMBI; 1:1 mixture of 4-ethyl and 5-methyl isomers).

Basis for delineation of the DNEL:

Study (rat study):

Repeated dose study

rat, male, female,

subacute oral gavage study for 28 days

rat: 0 (control), 4, 20 100 mg/kg bw/day – male, female

via gavage

Effects, NOAEL:

NOAEL = 20 mg/kg bw/day (male + female rats)*

*See Attachment 1

Effects:

Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MB2 at doses of 20 and 100 mg/kg bw/day. Male rats administered 100 mg/kg bw/day MB2 exhibited a 1.8 fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MB2/kg bw/day exhibited significant increases of liver and kidney but not thyroid weights, and serum cholesterol level.

1.) Long-term toxicity – systemic effects (worker)

Long-term oral or dermal route-systemic effects (worker) using default extrapolation factors:

NOAEL(rat, male) from a subacute toxicity study: 20 mg/kg bw/day

Penetration oral compared to dermal (both assumed 100%) 1

For interspecies rat vs. human: 4 (default value ECHA)

For remaining interspecies differences: 2.5 (default value ECHA)

For intraspecies differences in workers: 5 (default value ECHA)

For extrapolation of exposure duration subacute to chronic: 6 (default value ECHA)

For reliability of dose-response: 1

For quality of whole database: 1

Overall factor: 300

Worker DNEL long-term for oral or dermal route-systemic: 0.093 mg/kg bw/day

Long-term inhalation route-systemic effects (worker):

NOAEL(rat) from a subacute oral toxicity study: 20 mg/kg bw/day

Oral absorption = inhalatory absorption = 100*

*See Attachment 2

Correction of the starting point according TGD Figure R.8-3:

Corrected inhalatory NOAEC = Oral NOAEL (20 mg/kg) x 1/0.38 m³/kg x 6.7 m³/10m³ x 7/5

=> NOAEC worker = 49.4 mg/m³

For interspecies differences rat vs. human: 1 (according TGD Table

R.8-4. already covered by correction of starting point)

For remaining interspecies differences: 2.5 (default value ECHA)

For intraspecies differences in workers: 5 (default value ECHA)

For extrapolation of exposure duration subacute to chronic: 6 (default value ECHA)

For reliability of dose-response: 1

For quality of whole database: 1

Overall factor: 75

Worker DNEL long-term for inhalation exposure: 0.66 mg/m³

2.) Short-term toxicity – systemic effects (workers)

Concerning the systemic effects an exceeding factor of 5 based on the DNEL for long term exposure seems justified.

Worker DNEL short-term for oral or dermal route-systemic: 0.46 mg/kg bw/day

Worker DNEL short-term for inhalation exposure: 3.3 mg/m³

Conclusion (systemic effects):

Worker DNEL long-term for oral or dermal route-systemic: 0.093 mg/kg bw/day

Worker DNEL long-term for inhalation exposure: 0.66 mg/m³

Worker DNEL short-term for oral or dermal route-systemic: 0.46 mg/kg bw/day

Worker DNEL short-term for inhalation exposure: 3.3 mg/m³

3.) Reproductive Toxicity – systemic effects (worker)

No reproductive toxicity study is available for 2-mercaptomethylbenzimidazole MB2 (CAS 53988-10-6). Based on the instability of ZMB2 in aqueous solution, i.e. the quantitative dissociation within seconds, a read-across from MB2 and vice versa is fully valid and justified (ECHA communication number: TPE-C-2114492370-50-01/F).

In an extended one generation reproductive toxicity study according to OECD TG 443, Vulkanox ZMB2 was administered orally, by gavage, to CD rats at dose levels of 5, 15 or 40 mg/kg bw/day. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, developmental neurotoxicity and developmental immunotoxicity assessments were included, along with an evaluation of the maturing reproductive tract and its integrity and function.

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 15 mg/kg/day due to the incidences of prolonged parturition/dystocia in females of both generations receiving 40 mg/kg/day.

Aside from the above mentioned instances of prolonged parturition/dystocia among females at 40 mg/kg/day, increased incidences of liver hypertrophy, thyroid gland hypertrophy and involution/atrophy of the thymus were observed at 40 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 15 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 15 mg/kg/day due to reduced early post-partum survival at 40 mg/kg/day in both generations.

There was no evidence of developmental neurotoxicity or developmental immunotoxicity on this study, therefore the NOAEL for these endpoints was concluded to be 40 mg/kg/day.

Therefore, for worker the DNEL long-term is 0.42 mg/kg/d (oral) and 1.48 mg/m³ (inhalation).

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) four groups of 20 females received Vulkanox ZMB2 at doses of 8, 25 or 70 mg/kg bw/day by oral gavage administration, from Day 6 to 19 after mating, at a volume dose of 5mL/kg body weight. A similarly constituted Control group received the vehicle, dried corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg bw/day.

The derivation of a separate DNEL for reproductive toxicity is not necessary, because the DNEL for repeated dose toxicity covers reproductive toxicity.

4. Long-term and short-term dermal or inhalation route - local effects (worker)

In rabbits, MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) was not irritating to the skin, and not irritating to the eyes.

5. Sensitization

MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) was not sensitising in a LLNA.

MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) is not sensitizing and not irritating to the skin and eyes. Therefore, no hazard is identified for local effects is not applicable.

Conclusion (systemic and local effects - worker):

Route of exposure DNEL: local effect DNEL systemic effect

Oral (long term) no hazard identified 0.093 mg/kg bw/day

Oral (short term) no hazard identified 0.46 mg/kg bw/day

Dermal (long term) no hazard identified 0.093 mg/kg bw/day

Dermal (short term) no hazard identified 0.46 mg/kg bw/day

Inhalation (long term) no hazard identified 0.66 mg/m³

Inhalation (short term) no hazard identified 3.3 mg/m³

Attachment 1:

MB2 CAS 53988-10-6_DNEL_NOAEL starting point

In a subacute oral toxicity study with MB2 (2-mercaptomethylbenzimidazole; also named MMBI; 1:1 mixture of 4-ethyl and 5-methyl isomers) male and female rats were treated by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg bw/day for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times, and micronuclei induction in bone marrow erytropoetic cells, and histopathology were examined (Saitoh et al., 1999).

“Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MMBIs at doses of 20 and 100 mg/kg. Males rats administered 100 mg/kg MMBIs exhibited a 1.8 fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MMBIs/kg exhibited significant increases of liver and kidney weights, and serum cholesterol level.

No-observed-effect levels for male and female rats were found to be 4 and 20 mg/kg, respectively, in this subacute oral toxicity study”.

Organ weight findings male rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
Body weight [g]	212 + 9	210+9	206+14	195+8	252+17	244+14
Absolute organ weight
Lung [g]	0.75 + 0.03	0.76 + 0.04	0.82 + 0.04	0.80 + 0.07	0.84 + 0.07	0.86 + 0.08
Liver [g]	6.38 + 0.73	6.26 + 0.55	6.96 + 0.76	7.49 + 0.55**	7.48 + 0.86	7.57 + 0.77
Kidney [g]	1.45 + 0.06	1.42 + 0.10	1.53 + 0.09	1.51 + 0.13	1.58 + 0.07	1.55 + 0.16
Thyroid [g]	10.6 + 1.2	12.4 + 1.4	12.3 + 1.7	17.3 + 2.6**	13.4 + 2.5	14.5 + 0.7
Relative organ weight
Lung [g%]	0.35 + 0.03	0.36 + 0.02	0.40 + 0.02**	0.41 + 0.02**	0.33 + 0.01	0.35 + 0.02
Liver [g%]	3.00 + 0.23	2.97 + 0.14	3.37 + 0.03**	3.85 + 0.18**	2.96 + 0.19	3.11+ 0.16
Kidney [g%]	0.68 + 0.02	0.67 + 0.01	0.74 + 0.03**	0.77 + 0.04**	0.63 + 0.02	0.64 + 0.04
Thyroid [mg%]	4.98 + 0.53	5.92 + 0.89	6.00 + 1.10	8.86 + 1.23**	5.30 + 0.73	5.96 + 0.32

Organ weight findings for male rats after 28 days of treatment with MB2 and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Relative lung, liver and kidney weights were significantly increased in male rats treated with 20 mg/kg bw/day. However, absolute lung, liver and kidney weights in male rats were not increased significantly in male rats treated with 20 mg/kg bw/day. The increase in relative organ weights is slight and not evident in the recovery groups at 100 mg/kg bw/day. There is no correlate with histopathology finding in lung, liver and kidneys up to the highest dose of 100 mg/kg bw/day. The effects seen in relative organ weights at 20 mg/kg bw/day are regarded as secondary effects due to the slightly (2.4%) decreased body weight.

Taking the above information into account, the relative organ weight effects at 20 mg/kg bw/day are not considered adverse and the NOAEL for male rats is concluded to be 20 mg/kg bw/day.

In female rats relative and absolute organ weights of lung, liver and kidney were not increased at a dose of 20 mg/kg bw/day.

Therefore, the NOAEL for males and females is 20 mg/kg bw/day.

Organ weight findings female rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
Body weight [g]	132 + 8	135 + 9	128 + 11	133 + 7	142 + 16	141 + 9
Absolute organ weight
Lung [g]	0.65 + 0.15	0.61 + 0.06	0.61 + 0.01	0.70 + 0.11	0.61 + 0.11	0.63 + 0.07
Liver [g]	3.70 + 0.31	3.85 + 0.27	3.97 + 0.56	5.10 + 0.96**	3.55 + 0.50	3.73 + 0.29
Kidney [g]	0.96 + 0.04	1.00 + 0.04	0.99 + 0.07	1.12 + 0.12**	0.92 + 0.10	0.98 + 0.10
Thyroid [g]	11.50 + 2.32	9.28 + 1.41	10.94 + 1.63	11.18 + 1.76	12.10 + 1.84	12.42 + 1.62
Relative organ weight
Lung [g%]	0.49 + 0.12	0.45 + 0.02	0.48 + 0.04	0.52 + 0.06	0.43 + 0.08	0.44 + 0.07
Liver [g%]	2.81 + 0.08	2.85 + 0.03	3.10 + 0.42	3.82 + 0.73**	2.49 + 0.18	2.65 + 0.16
Kidney [g%]	0.74 + 0.03	0.74 + 0.04	0.77 + 0.03	0.84 + 0.09**	0.65 + 0.04	0.69 + 0.05
Thyroid [mg%]	8.78 + 1.91	6.90 + 1.16	8.50 + 0.76	8.38 + 1.23	8.50 + 0.87	8.82 + 1.35

Organ weight findings for female rats after 28 days of treatment with MB2 and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Histopathological findings (excerpt, Saitoh et al., 1999)

	Treatment								Recovery
Sex	Male				Female				Male		Female
Dose [mg/kg] No. of animals examined	0 5	4 5	20 5	100 5	0 5	4 5	20 5	100 5	0 5	100 5	0 5	100 5
Lung
Thickening of alveolar wall	1	0	2	0	0	1	1	0	0	0	1	0
Foamy cell infiltration	0	1	0	0	0	0	0	0	0	0	0	0
Perivascular oedema	1	1	0	0	1	1	0	0	1	0	0	0
Liver
Hepatocyte vacuolisation	0	0	1	1	0	0	0	0	0	0	0	0
Microgranuloma formation	2	0	1	0	0	0	0	0	0	0	0	0
Hepatocyte swelling	0	0	0	0	0	0	0	5	0	0	0	0
Kidney
Eosinophilic bodies	5	5	5	5	0	0	0	0	5	5	0	0

Histopathology findings for rats after 28 days of treatment with MB2 and the 14-day recovery period

Biochemical findings in male rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
PL (phospholipid) [mg/dl]	115 + 11	115 + 6	130 + 8	134 + 11**	136 + 17	134 + 5
T-CHO (total cholesterol) [mg/dl]	48 + 5	49 + 5	63 + 5**	74 + 6**	55 + 7	58 + 3

Biochemical findings for male rats after 28 days of treatment of 2-mercaptomethylbenzimidazoles and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Biochemical findings in female rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
PL (phospholipid) [mg/dl]	133 + 19	135 + 9	144 + 10	152 + 22	162 + 20	165 + 10
T-CHO (total cholesterol) [mg/dl]	59 + 11	60 + 8	71 + 3	88 + 16**	81 + 10	79 + 4

Biochemical findings for female rats after 28 days of treatment with MB2 and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

The PL (phospholipid) level was statistically increased in male rats only at 100 mg/kg bw/day and no effect was observed in female rats at any dose.

A slight but significant higher T-CHO (total cholesterol) level was observed in male rats at doses of 20 mg/kg bw/day and 100 mg/kg bw/day. The increase was approx. 25% and 50 %, respectively. In female rats the T-CHO level was increased at 100 mg/kg bw/day (approx. 25%) only andno significant increase in T-CHO level was observed in female rats at 20 mg/kg bw/day and below.Increased cholesterol levels in this study are accompanied by an increase of liver weights in male and female rats. The increase of cholesterol is interconnected with an increase of the liver weights. The increase of the liver weights is dose dependent and a result of the exposure of the rats to the test substance. Liver weight changes in repeated dose studies in mammalians are often caused by liver enzyme induction. An important function of the liver is to produce and clear cholesterol in the body. If the liver is not working properly, it can cause cholesterol to build up in the body.

In the subacute study by Saitoh et al. (1999), the T-CHO values were only slightly increased in male rats at 20 mg/kg bw/day. The effect was reversible in the recovery group at 100 mg/kg bw/day; the T-CHO values in the control and the 100 mg/kg bw/day groups were similar and there were no significant differences in liver or thyroid weights in the control and the 100 mg/kg bw/day groups. These data indicate, that the elevated cholesterol levels at 20 mg/kg bw/day are adaptive and should be considered non-adverse.

Conclusion:

The NOEL (no-observed-effect level) for male and female rats are 4 and 20 mg/kg bw/day, respectively.

The NOAEL (no-observed-adverse-effect level) for male and female rats are concluded to be 20 mg/kg bw/day.

Attachment 2:

MB2 CAS 53988-10-6_ Assessment factors for oral/inhalatory absorption

For the derivation of a systemic DNEL for MB2 (2-mercaptomethylbenzimidazole, also named MMBI; 1:1 mixture of 4-ethyl and 5-methyl isomers) a subacute oral toxicity study is available:

In a subacute oral toxicity study with MB2 male and female rats were treated by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg bw/day for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times, and micronuclei induction in bone marrow erytropoetic cells, and histopathology were examined (Saitoh et al., 1999).

The NOAEL (no-observed-adverse-effect level) for male and female rats are concluded to be 20 mg/kg bw/day; see separate justification for NOAEL starting point.

No repeated dose inhalation or repeated dose dermal study is available. Therefore, a route-to-route extrapolation is conducted to derive systemic DENLs for these routes of exposure.

In the ECHA ‘Guidance on information requirements and chemical safety assessment, Chapter R.8: Characterisation of dose [concentration] response for human health’, ECHA provides extensive guidance on the application of assessment factors to address the differences between an experiment and a human situation, and the uncertainties associated with these differences. A set of default assessment factors is defined in ECHA's guidance.

“Default absorption values have been proposed for the different routes of exposure (see Section R.7.12. on toxicokinetics), but substance-specific data on absorption via the different routes are to be preferred. Such information may for instance be generated based on considerations of the chemical structure.

In the absence of these data for both the starting route and the end route (the route to which the extrapolation is being made), worst case assumptions have to be made. Worst case in this context will be obtained assuming a limited absorption for the starting route, leading to a low (conservative) internal NOAEL. To secure a conservative external NOAEL a maximum absorption should there after be assumed for the end route, leading to a low external NOAEL. It is proposed, thus, in the absence of route-specific information on the starting route, to include a default factor of 2 (i.e. the absorption percentage for the starting route is half that of the end route) in the case of oral-to-inhalation extrapolation. The inclusion of this factor 2 means for example that 50% (instead of 100%) absorption is assumed for oral absorption, and 100% for inhalation. Note that if data on the starting route (oral) are available these should be used, but for the end route (inhalation), the worst case inhalation absorption should still be assumed (i.e. 100%)”.

MB2 (2-mercaptomethylbenzimidazole, MMBI = 1:1 mixture of 4-ethyl and 5-methyl isomers) is structurally very similar to MBI (mercaptobenzimidazole).

MBI (mercaptobenzimidazole)

MB2 (2-mercaptomethylbenzimidazole) =

MMBI = 1:1 mixture of 4-ethyl and 5-methyl isomers)

In a single dose toxicokinetic study the excretion of [14C]-labeled MBI in male Fisher-344 rats dosed orally (49 or 0.5 mg/kg) or intravenously (0.5 mg/kg) was determined (El Dareer et al., (1984)).

The following excretion of radioactivity from [14C] MBI for rats dosed orally or intravenously was found:

	Percent of dose
Sample collected	Oral high dose (49 mg/kg)	Oral low dose (0.5 mg/kg)	Intravenous dose (0.5 mg/kg)
Urine:
24 h	40.1 + 6.9	66.0 + 20.1	48.7 + 9.3
48 h	25.3 + 7.4	2.34 + 0.41	2.59 + 1.76
72 h	1.34 + 0.42	0.488 + 0.165	0.458 + 0.178
Total urine	66.7 + 12.9	68.8 + 20.5	51.7 + 7.5
Faeces:
24 h	10.3 + 8.6	21.1 + 13.9	26.2 + 6.0
48 h	6.66 + 1.63	3.38 + 0.33	4.38 + 2.18
72 h	1.78 + 0.48	0.972 + 0.233	0.509 + 0.135
Total faeces	18.8 + 9.0	25.4 + 13.8	31.1 + 7.7
Total recovery (72 h)	88.7 + 3.9	95.5 + 11.7	84.6 + 1.0

Excretion of radioactivity after oral or IV dosing of rats with [14C] MBI

Based on the data of the toxicokinetic excretion study, it can be concluded that oral doses of 0.5 and 49 mg/kg were nearly completely absorbed after oral administration of the test substance MBI.

The amount of MBI in the faeces of 31.1 + 7.7% after intravenous application was higher than the amount in the faeces after oral administration (oral high dose: 18.8 + 9.0%, oral low dose: 25.4 + 13.8%).

The amount found in the faeces after oral application is therefore not the result of a restricted absorption, instead these results indicate that systemically available MBI is partly excreted in the bile and finally excreted via faeces and consequently not an indication for a poor or low absorption of MBI from the gastrointestinal tract.

In a comparative toxicokinetic study by Sakemi K et al., (1999), the authors reported that single oral administration toxicity studies of MBI and MMBI showed that they have similar acute toxicity. Their LD50 values are reported to be 300 and 330 mg/kg, respectively.

No toxic signs were observed by single oral administrations of MBI or MMBI at 50 mg/kg or less. At 250 mg/kg, both MBI and MMBI caused the decrease in locomotor activity, ataxic and paralytic gait, lacrimation, gasping and coma. Five out of six rats orally given 250 mg/kg of MBI died from 12 hr to 50 hr after treatment and one rat survived. Five out of six rats orally given 250 mg/kg of MMBI died within 26 hr after treatment and one rat survived. At autopsy, there were no toxicological findings at any dose of MBI. Apparent enlargement of the liver with a faded colour was observed at 250 mg/kg of MMBI. Single i.p. administration of MBI or MMBI at 50 mg/kg caused the decrease in locomotor activity from 30 min for 3 – 4 hr.

According to the authors of the study, MBI and MMBI have similar serum Cmax values at early hr at the same administration doses and therefore cause the similar acute toxicities. Although their Cmax values were similar, the slower disappearance of MBI in the serum resulted in larger AUC values for MBI than for MMBI.

However, the slower clearance is a result of the metabolic elimination in rats and not due to differences in their absorption rates.

Conclusion:

For MBI and MMBI a single oral administration resulted in similar LD50 values of 300 and 330 mg/kg, respectively. The same toxicological signs were reported for both compounds at identical applications doses. The serum Cmax values at early ours were similar. Based on these results similar absorption rates of MBI and MMBI were assumed.

As demonstrated in the toxicokinetic study with [14C] labelled MBI, oral doses of 0.5 and 49 mg/kg were nearly completely absorbed after oral administration of the test substance MBI. As a result of the acute toxicity studies an absorption rate of nearly 100% can also be assumed for MB2.

Therefore, it is justified, that the oral administration is high (100%) and consequently similar to the conservative default absorption by inhalation defined in the REACH guidance documents (100%).

For the route-to route derivation oral to inhalation no correction factor due to difference in absorption is necessary in the DNEL derivation for workers and the general population.

The equations for the corrected inhalation NOAEC for MB2 (CAS 53988-10-6) are as follows:

Worker:

corrected inhalatory NOAEC = oral NOAEL x 1/(0.38 ) x (ABS oral-rat)/(ABS inh-rat) x 7/5 x 6.7/10

ABS oral-rat = ABS inh-rat = 100%

7/5: rats were exposed for 28 consecutive days, workers are exposed for 5 days

General population:

corrected inhalatory NOAEC = oral NOAEL x 1/(1.15 ) x (ABS oral-rat)/(ABS inh-rat)

ABS oral-rat = ABS inh-rat = 100%

References:

Saitoh M et al., Toxicity study of a Rubber antioxidant, mixture of 2-mercaptomethylbenzimidazoles, by repeated oral administration to rats, Food and Chemical Toxicology 37, 777-787 (1999)

Sakemi K et al., Comparative toxicokinetik study of rubber oxidants, 2-mercaptobenzimidazole and 2-mercaptomethylbenzimidazole, by single oral administration in rats, The Journal of Toxicological Sciences, 24, 399-405 (1999)

El Dareer SM et al., Disposition of 2-mercaptobenzimidazole in rats dosed orally or intravenously, Journal of Toxicology and Environmental Health, 14, 595-604 (1984)

Thyssen J, Untersuchungen zur Haut- und Schleimhautvertraeglichkeit - Versuchsprodukt KA 9059 = Vulkanox MB-2/MG, Bayer - Institut fuer Toxikologie Wuppertal-Elberfeld, 1977

Thyssen J, Untersuchungen zur Haut- und Schleimhautvertraeglichkeit - ZMB-2, Bayer - Institut fuer Toxikologie Wuppertal-Elberfeld, 1979

Leidenfrost P, 1,3-Dihydro-4(or 5)-methyl-2H-benzimidaz9ole-2-thione Local Lymph node assay in mice (LLNA/IMDS), Bayer Pharma AG GDD-GED-Toxicology, 42096 Wuppertal, Germany, 2011

Stannard D, Vulkanox ZMB2: Extended One Generation Reproductive Toxicity Study in the CD Rat by Oral Gavage Administration, Covance CRS Limited, Report no. QG31PJ, 19 January 2020

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.11 mg/m³

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

Overall assessment factor (AF):

150

Dose descriptor starting point:

NOAEL

Value:

20 mg/kg bw/day

Modified dose descriptor starting point:

NOAEC

Value:

17.4 mg/m³

AF for dose response relationship:

1

AF for differences in duration of exposure:

6

Justification:

default value (ECHA)

AF for interspecies differences (allometric scaling):

1

Justification:

default value (ECHA)

AF for other interspecies differences:

2.5

Justification:

default value (ECHA)

AF for intraspecies differences:

10

Justification:

default value (ECHA)

AF for the quality of the whole database:

1

Justification:

see 'Discussion'

AF for remaining uncertainties:

1

Justification:

see 'Discussion'

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.55 mg/m³

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

DNEL extrapolated from long term DNEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.033 mg/kg bw/day

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

Overall assessment factor (AF):

600

Dose descriptor starting point:

NOAEL

Value:

20 mg/kg bw/day

AF for dose response relationship:

1

Justification:

see 'Discussion'

AF for differences in duration of exposure:

6

Justification:

default value (ECHA)

AF for interspecies differences (allometric scaling):

4

Justification:

default value (ECHA)

AF for other interspecies differences:

2.5

Justification:

default value (ECHA)

AF for intraspecies differences:

10

Justification:

default value (ECHA)

AF for the quality of the whole database:

1

Justification:

see 'Discussion'

AF for remaining uncertainties:

1

Justification:

see 'Discussion'

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.16 mg/kg bw/day

DNEL extrapolated from long term DNEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.033 mg/kg bw/day

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

Overall assessment factor (AF):

600

Dose descriptor starting point:

NOAEL

Value:

20 mg/kg bw/day

AF for dose response relationship:

1

Justification:

see 'Discussion'

AF for differences in duration of exposure:

6

Justification:

default value (ECHA)

AF for interspecies differences (allometric scaling):

4

Justification:

default value (ECHA)

AF for other interspecies differences:

2.5

Justification:

default value (ECHA)

AF for intraspecies differences:

10

Justification:

default value (ECHA)

AF for the quality of the whole database:

1

Justification:

see 'Discussion'

AF for remaining uncertainties:

1

Justification:

see 'Discussion'

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

0.16 mg/kg bw/day

Route of original study:

Oral

DNEL derivation method:

ECHA REACH Guidance

DNEL extrapolated from long term DNEL

Hazard assessment conclusion:

no hazard identified

2-Mercaptomethylbenzimidazole MB2 (CAS 53988-10-6)

and its and Na+ salt (CAS 75045-07-7)

DNELs (general population)

Repeated dose toxicity

A subacute oral gavage study rats was evaluated for the derivation of DNELs of MB2 (2-mercapto-methylbenzimidazole; also named MMBI; 1:1 mixture of 4-ethyl and 5-methyl isomers).

Basis for delineation of the DNEL:

Study (rat study):

Repeated dose study

rat, male, female,

subacute oral gavage study for 28 days

rat: 0 (control), 4, 20 100 mg/kg bw/d – male, female

via gavage

Effects, NOAEL:

NOAEL = 20 mg/kg bw/d (male + female rats)*

*See Attachment 1

Effects:

Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MB2 at doses of 20 and 100 mg/kg bw/day. Male rats administered 100 mg/kg bw/day MB2 exhibited a 1.8 fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MB2/kg bw/day exhibited significant increases of liver and kidney but not thyroid weights, and serum cholesterol level.

Reference:

Saitoh M, Umemura T, Kawasaki Y, Momma Y, Matsushima Y, Sakemi K, Isama K, Kitajima S, Ogawa Y, Hasegawa R, Suzuki T, Hayashi M, Inoue T, Ohno Y, Sofuni T, Kurokawa Y, Tsuda M

Toxicity study of a rubber antioxidant, mixture of 2-mercaptomethylbenzimidazoles, by repeated oral administration to rats

Food Chem Toxicol 37, 777-787 (1999)

1.) Long-term tocixity – systemic effects (general population)

Long-term oral or dermal route-systemic effects (general population) using default extrapolation factors:

NOEL(rat, male) from a subacute toxicity study: 20 mg/kg bw/day

For interspecies rat vs. human: 4 (default value ECHA)

For remaining interspecies differences: 2.5 (default value ECHA)

For intraspecies differences in general population: 10 (default value ECHA)

For extrapolation of exposure duration subacute to chronic: 6 (default value ECHA)

For reliability of dose-response: 1

For quality of whole database: 1

Overall factor: 600

General population DNEL long-term for oral or dermal route-systemic: 0.033 mg/kg bw/day

Long-term inhalation route-systemic effects (general population):

NOEL(rat) from a subacute oral toxicity study: 20 mg/kg bw/day

Oral absorption = inhalatory absorption = 100*

*See Attachment 2

Correction of the starting point according TGD Figure R.8-3:

Corrected inhalatory NOAEC = Oral NOAEL (20 mg/kg) x 1/1.15 m³/kg x 1.0

=> NOAEC general population = 17.4 mg/m³

For interspecies differences rat vs. human: 1 (according TGD Table

R.8-4. already covered by correction of starting point)

For remaining interspecies differences: 2.5 (default value ECHA)

For intraspecies differences in general population: 10 (default value ECHA)

For extrapolation of exposure duration subacute to chronic: 6 (default value ECHA)

For reliability of dose-response: 1

For quality of whole database: 1

Overall factor: 150

General population DNEL long-term for inhalation exposure: 0.11 mg/m³

2.) Short-term toxicity – systemic effects (general population)

Concerning the systemic effects an exceeding factor of 5 based on the DNEL for long term exposure seems justified.

General population DNEL short-term for oral or dermal route-systemic: 0.16 mg/kg bw/day

General population DNEL short-term for inhalation exposure: 0.55 mg/m³

Conclusion (systemic effects):

General population DNEL long-term for oral or dermal route-systemic: 0.033 mg/kg bw/day

General population DNEL long-term for inhalation exposure: 0.11 mg/m³

General population DNEL short-term for oral or dermal route-systemic: 0.16 mg/kg bw/day

General population DNEL short-term for inhalation exposure: 0.55 mg/m³

3.) Reproductive Toxicity – systemic effects (general population)

No reproductive toxicity study is available for MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6). Based on the instability of ZMB2 in aqueous solution, i.e. the quantitative dissociation within seconds, a read-across from MB2 and vice versa is fully valid and justified (ECHA communication number: TPE-C-2114492370-50-01/F).

In an extended one generation reproductive toxicity study according to OECD TG 443, Vulkanox ZMB2 was administered orally, by gavage, to CD rats at dose levels of 5, 15 or 40 mg/kg bw/day. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, developmental neurotoxicity and developmental immunotoxicity assessments were included, along with an evaluation of the maturing reproductive tract and its integrity and function.

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 15 mg/kg/day due to the incidences of prolonged parturition/dystocia in females of both generations receiving 40 mg/kg/day.

Aside from the above mentioned instances of prolonged parturition/dystocia among females at 40 mg/kg/day, increased incidences of liver hypertrophy, thyroid gland hypertrophy and involution/atrophy of the thymus were observed at 40 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 15 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 15 mg/kg/day due to reduced early post-partum survival at 40 mg/kg/day in both generations.

There was no evidence of developmental neurotoxicity or developmental immunotoxicity on this study, therefore the NOAEL for these endpoints was concluded to be 40 mg/kg/day.

Therefore, for general population the DNEL long-term is 0.15 mg/kg bw/day (oral and dermal) and 0.26 mg/m³ (inhalation).

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) four groups of 20 females received Vulkanox ZMB2 at doses of 8, 25 or 70 mg/kg bw/day by oral gavage administration, from Day 6 to 19 after mating, at a volume dose of 5mL/kg body weight. A similarly constituted Control group received the vehicle, dried corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg bw/day.

The derivation of a separate DNEL for reproductive toxicity is not necessary, because the DNEL for repeated dose toxicity covers reproductive toxicity.

4. Long-term and short-term dermal or inhalation route - local effects (general population)

In rabbits, MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) was not irritating to the skin, and not irritating to the eyes.

5. Sensitization

2 MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) was not sensitising in a LLNA.

MB2 (2-mercaptomethylbenzimidazole, CAS 53988-10-6) is not sensitizing and not irritating to the skin and eyes. Therefore, no hazard is identified for local effects.

Conclusion (systemic and local effects – general population):

Route of exposure DNEL: local effect DNEL systemic effect

Oral (long term) no hazard identified 0.033 mg/kg bw/day

Oral (short term) no hazard identified 0.11 mg/kg bw/day

Dermal (long term) no hazard identified 0.033 mg/kg bw/day

Dermal (short term) no hazard identified 0.11 mg/kg bw/day

Inhalation (long term) no hazard identified 0.16 mg/m³

Inhalation (short term) no hazard identified 0.55 mg/m³

Attachment 1:

MB2 CAS 53988-10-6_DNEL_NOAEL starting point

In a subacute oral toxicity study with MB2 (2-mercaptomethylbenzimidazole; also named MMBI; 1:1 mixture of 4-ethyl and 5-methyl isomers) male and female rats were treated by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg bw/day for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times, and micronuclei induction in bone marrow erytropoetic cells, and histopathology were examined (Saitoh et al., 1999).

“Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MMBIs at doses of 20 and 100 mg/kg. Males rats administered 100 mg/kg MMBIs exhibited a 1.8 fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MMBIs/kg exhibited significant increases of liver and kidney weights, and serum cholesterol level.

No-observed-effect levels for male and female rats were found to be 4 and 20 mg/kg, respectively, in this subacute oral toxicity study”.

Organ weight findings male rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
Body weight [g]	212+9	210+9	206+14	195+8	252+17	244+14
Absolute organ weight
Lung [g]	0.75+0.03	0.76+0.04	0.82+0.04	0.80+0.07	0.84+0.07	0.86+0.08
Liver [g]	6.38+0.73	6.26+0.55	6.96+0.76	7.49+0.55**	7.48+0.86	7.57+0.77
Kidney [g]	1.45+0.06	1.42+0.10	1.53+0.09	1.51+0.13	1.58+0.07	1.55+0.16
Thyroid [g]	10.6+1.2	12.4+1.4	12.3+1.7	17.3+2.6**	13.4+2.5	14.5+0.7
Relative organ weight
Lung [g%]	0.35+0.03	0.36+0.02	0.40+0.02**	0.41+0.02**	0.33+0.01	0.35+0.02
Liver [g%]	3.00+0.23	2.97+0.14	3.37+0.03**	3.85+0.18**	2.96+0.19	3.11+0.16
Kidney [g%]	0.68+0.02	0.67+0.01	0.74+0.03**	0.77+0.04**	0.63+0.02	0.64+0.04
Thyroid [mg%]	4.98+0.53	5.92+0.89	6.00+1.10	8.86+1.23**	5.30+0.73	5.96+0.32

Organ weight findings for male rats after 28 days of treatment with MB2 and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Relative lung, liver and kidney weights were significantly increased in male rats treated with 20 mg/kg bw/day. However, absolute lung, liver and kidney weights in male rats were not increased significantly in male rats treated with 20 mg/kg bw/day. The increase in relative organ weights is slight and not evident in the recovery groups at 100 mg/kg bw/day. There is no correlate with histopathology finding in lung, liver and kidneys up to the highest dose of 100 mg/kg bw/day. The effects seen in relative organ weights at 20 mg/kg bw/day are regarded as secondary effects due to the slightly (2.4%) decreased body weight.

Taking the above information into account, the relative organ weight effects at 20 mg/kg bw/day are not considered adverse and the NOAEL for male rats is concluded to be 20 mg/kg bw/day.

In female rats relative and absolute organ weights of lung, liver and kidney were not increased at a dose of 20 mg/kg bw/day.

Therefore, the NOAEL for males and females is 20 mg/kg bw/day.

Organ weight findings female rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
Body weight [g]	132+8	135+9	128+11	133+7	142+16	141+9
Absolute organ weight
Lung [g]	0.65+0.15	0.61+0.06	0.61+0.01	0.70+0.11	0.61+0.11	0.63+0.07
Liver [g]	3.70+0.31	3.85+0.27	3.97+0.56	5.10+0.96**	3.55+0.50	3.73+0.29
Kidney [g]	0.96+0.04	1.00+0.04	0.99+0.07	1.12+0.12**	0.92 + 0.10	0.98+0.10
Thyroid [g]	11.50+2.32	9.28+1.41	10.94+1.63	11.18+1.76	12.10+1.84	12.42+1.62
Relative organ weight
Lung [g%]	0.49+0.12	0.45+0.02	0.48+0.04	0.52+0.06	0.43+0.08	0.44+0.07
Liver [g%]	2.81+0.08	2.85+0.03	3.10+0.42	3.82+0.73**	2.49+0.18	2.65+0.16
Kidney [g%]	0.74+0.03	0.74+0.04	0.77+0.03	0.84+0.09**	0.65+0.04	0.69+0.05
Thyroid [mg%]	8.78+1.91	6.90+1.16	8.50 + 0.76	8.38+1.23	8.50+0.87	8.82+1.35

Organ weight findings for female rats after 28 days of treatment with MB2 and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Histopathological findings (excerpt, Saitoh et al., 1999)

	Treatment								Recovery
Sex	Male				Female				Male		Female
Dose [mg/kg] No. of animals examined	0 5	4 5	20 5	100 5	0 5	4 5	20 5	100 5	0 5	100 5	0 5	100 5
Lung
Thickening of alveolar wall	1	0	2	0	0	1	1	0	0	0	1	0
Foamy cell infiltration	0	1	0	0	0	0	0	0	0	0	0	0
Perivascular oedema	1	1	0	0	1	1	0	0	1	0	0	0
Liver
Hepatocyte vacuolisation	0	0	1	1	0	0	0	0	0	0	0	0
Microgranuloma formation	2	0	1	0	0	0	0	0	0	0	0	0
Hepatocyte swelling	0	0	0	0	0	0	0	5	0	0	0	0
Kidney
Eosinophilic bodies	5	5	5	5	0	0	0	0	5	5	0	0

Histopathology findings for rats after 28 days of treatment with MB2 and the 14-day recovery period

Biochemical findings in male rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
PL (phospholipid) [mg/dl]	115+11	115+6	130+8	134+11**	136+17	134+5
T-CHO (total cholesterol) [mg/dl]	48+5	49+5	63+5**	74+6**	55+7	58+3

Biochemical findings for male rats after 28 days of treatment of 2-mercaptomethylbenzimidazoles and the 14-day recovery period

Data are mean + SD values

* and ** show significant differences from controls at P < 0.05 and P < 0.01, respectively

Biochemical findings in female rats (excerpt, Saitoh et al., 1999)

	Treatment				Recovery
Groups (dose) No. of rats	Control 5	4 mg/kg 5	20 mg/kg 5	100 mg/kg 5	Control 5	100 mg/kg 5
PL (phospholipid) [mg/dl]	133+19	135+9	144+10	152+22	162+20	165+10
T-CHO (total cholesterol) [mg/dl]	59+11	60+8	71+3	88+16**	81+10	79+4