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1.DOSE-RESPONSE ANALYSIS

1.1. Inhalation Reference Concentration for Effects other than Cancer for 1,2,3-TMB 

1.1.1. Identification of Candidate Principal Studies and Critical Effects for 1,2,3-TMB 

The nervous, hematological, and respiratory systems are the primary targets of inhaled 

1,2,3-TMB in humans and experimental animals, and effects in these systems have been 

identified as hazards following inhalation exposure to 1,2,3-TMB. Human data are 

preferred over animal data for deriving reference values when possible because the use of 

human data is more relevant in the assessment of human health and avoids the uncertainty 

associated with interspecies extrapolation introduced when animal data serve as the basis 

for the RfC. In this case, literature exists on the noncancer effects of 1,2,3-TMB exposure in 

humans, including neurological, hematological, and respiratory toxicities; however, no 

human studies are available that would allow for dose-response analysis. The human 

studies evaluated TMB exposures occurring as a complex solvent or VOC mixtures, and this 

consideration along with other uncertainties including high imprecision in effect measures 

due to low statistical power, lack of quantitative exposure assessment, and lack of control 

for co-exposures, limits their utility in derivation of quantitative human health toxicity 

values. However, these studies provide supportive evidence for the neurological, 

hematological, and respiratory toxicity of TMB isomers in humans. 

Several studies investigating 1,2,3-TMB noncancer effects in experimental animal models 

were identified in the literature. No chronic studies were available, although several acute, 

short-term, and subchronic studies were identified. 1,2,3-TMB-induced toxicity was 

observed across several organ systems in two subchronic studies by Korsak et al. (2000b)

and Korsak and Rydzyński (1996). These were the only subchronic studies identified in 

the peer-reviewed literature. Data from these studies pertaining to the primary hazards 

observed in humans and animals identified in Chapter 1, neurological, hematological, and 

respiratory toxicity, were considered as candidate critical effects for the purpose of 

determining the point of departure (POD) for derivation of the inhalation RfC for 1,2,3-

TMB. Neurotoxicity was also observed in both acute and short-term inhalation studies and 

respiratory toxicity was also observed in acute studies. However, the high exposure 

concentrations used in acute studies and the short exposure durations of both acute and 

short-term studies limit their applicability for quantitative assessment of chronic human 

health effects. Nevertheless, as with the human mixture studies, these studies provide 
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qualitative information regarding the consistency and coherency of these effects across the 

1,2,3-TMB database.

The two subchronic studies by Korsak et al. (2000b) and Korsak and Rydzyński (1996) are 

considered to be adequate for dose-response analysis. Both studies used rats, an 

appropriate laboratory animal species, and utilized appropriate sham-exposed controls. 

Animals were exposed to 1,2,3-TMB reported as > 97% pure (impurities not reported). 

The studies utilized an appropriate route (inhaled air) and duration (subchronic) of 

exposure. The studies used a reasonable range of appropriately-spaced exposure levels to 

facilitate dose-response analysis. An appropriate latency between exposure and 

development of toxicological outcomes was used, and the persistence of some outcomes 

after termination of exposure was investigated. Adequate numbers of animals per 

exposure group were used, and appropriate statistical tests including pair-wise and trend 

analyses were performed. 

With regard to reporting of exposure methodologies, Korsak et al. (2000b) reported actual 

exposure concentrations, as measured by gas chromatography, to be within 10% of target 

exposure concentrations. This increases the confidence in the overall evaluation and 

adequacy of this study. Although Korsak and Rydzyński (1996) do not report actual, 

measured exposure concentrations, this study uses the same exposure methodology as 

Korsak et al. (2000b); suggesting that it is likely that the actual exposure concentrations in 

this study are within 10% of target exposure concentrations. Target and actual 

concentrations for these studies are listed in Table 1-1. 

Table 1-1. Target and actual exposure concentrations used in BMD 

modeling of 1,2,3-TMB endpoints considered for the derivation of the 

RfC

Reference Species/

Sex

Target Exposure 

Concentration (mg/m3)

Actual Exposure 

Concentration (mg/m3)

Korsak and 

Rydzyński 

(1996)

Rat, male

123 n/a

492 n/a

1230 n/a

Korsak et al. 

(2000b)

Rat, male

123 128

492 523

1230 1269

Rat, female

123 128

492 523

1230 1269

n/a: not applicable

These subchronic studies examined 1,2,3-TMB-induced toxicity in multiple organ systems. 

Neurological, hematological, and respiratory endpoints that demonstrated statistically 
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significant pair-wise increases or decreases relative to control were considered for the 

derivation of the RfC for 1,2,3-TMB (Table 1-2). These endpoints included decreased pain 

sensitivity in male rats (Korsak and Rydzyński, 1996), and decreased RBCs and increased 

reticulocytes in male rats, decreased segmented neutrophils and increased lymphocytes in 

male and female rats, and increased inflammatory lung lesions in female rats (Korsak et al., 

2000b). Changes in liver organ weights and clinical chemistry parameters from Korsak et 

al. (2000b) were not further considered due to the lack of accompanying hepatocellular 

histopathological alterations in exposed animals. Changes in splenic organ weights were 

similarly not considered further due to a lack of any observed histopathological changes in 

that organ. Increases in reticulocytes in females were not further considered due to nonmonotonicity in response (i.e., non-significant increases in high exposure concentration 

animals). 

Table 1-2. Non-cancer endpoints resulting from subchronic inhalation 

exposure to 1,2,3-TMB considered for the derivation of the RfC

Endpoint Species/ 

Sex

Exposure Concentration (mg/m3)

0 123 492 1,230

Neurological Endpoints

Decreased pain sensitivity (measured as 

latency to paw-lick in seconds) Rat, male 9.7 ± 2.1a(n = 30)

11.8 ± 3.8d

(n = 20)

16.3 ± 6.3e

(n = 10)

17.3 ± 3.4c

(n = 10)

Hematological Endpoints

Decreased RBCs (106/cm3) Rat, male 9.49 ± 2.03

(n = 10)

10.2 ± 1.29

(n = 10)

10.11 ± 1.27

(n = 10)

8.05 ± 1.38b

(n = 10)

Decreased segmented neutrophils (%)

Rat, male 24.8 ± 4.5

(n = 10)

25.4 ± 5.8

(n = 10)

20.7 ± 5.8

(n = 10)

17.7 ± 8.3b

(n = 10)

Rat, female 23.1 ± 6.1

(n = 10)

19.7 ± 3.4

(n = 10)

16.4 ± 4.2b

(n = 10)

11.9 ± 7.1c

(n = 10)

Increased lymphocytes (%)

Rat, male 71.2 ± 5.0

(n = 10)

71.6 ± 6.8

(n = 10)

75.4 ± 4.7

(n = 10)

79.3 ± 78.0c

(n = 10)

Rat, female 73.2 ± 7.9

(n = 10)

77.5 ± 4.9

(n = 10)

80.4 ± 5.1

(n = 10)

84.0 ± 78.0c

(n = 10)

Increased reticulocytes (%) Rat, male 2.8 ± 1.3

(n = 10)

2.1 ± 1.7

(n = 10)

3.8 ± 2.1

(n = 10)

4.5 ± 1.8b

(n = 10)

Respiratory Endpoints

Increased inflammatory lung lesions Rat, female

e

(n = 10)

e

(n =10)

e

(n = 10)

e

(n = 10)

a Values are expressed as mean ± one standard deviation

bp < 0.05

c p < 0.01

d Level of significance not reported in Table 1 from Korsak and Rydzyński (1996), however the results of an adhoc t-test (performed by EPA) indicated significance at p < 0.01

e Incidences for exposure groups not reported; however, based on qualitative information reported in the study, 

i.e., that female rats exhibited a statistically significant increase in inflammatory lung lesions at 492 mg/m3. 

Adapted from Korsak et al., (2000b) and Korsak and Rydzyński (1996)
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Impaired neuromuscular function and coordination, measured as performance on rotarod 

apparatus, was also observed in rats exposed to 1,2,3-TMB. The use of rotarod data from 

Korsak and Rydzyński (1996) was initially considered for quantification of candidate RfC 

values for 1,2,3-TMB. However, upon critical evaluation of the exposure-response 

information in the study it was determined that the endpoint was reported in a manner 

that reduced the confidence in the observed effect levels. The critical limitation noted for 

these data related to the presentation of rotarod performance, which is best represented as 

a continuous variable, as opposed to a quantal variable such as that presented by Korsak 

and Rydzyński (1996). In contrast to the percent failures reported by the study authors, 

the most widely used and accepted measurement for rotarod performance in rodents is 

latency to fall from the rotating rod (Brooks and Dunnett, 2009; Kaspar et al., 2003; Bogo et 

al., 1981), typically with an arbitrary upper limit on the maximum latency allowed to 

prevent confounding by fatigue. Although the quantal percent failures data can provide 

useful information, these measures require an arbitrary selection of the length of time 

required for successful performance; there is no scientific consensus on an optimal time for 

this parameter. In addition, when identifying effect levels based on the data presented by 

Korsak and Rydzyński (1996), latencies on the rod of 1 and 119 seconds would be treated 

identically as failures when, in fact, they indicate very different levels of neurological 

dysfunction (Bogo et al., 1981). This adds uncertainty when trying to extrapolate to a 

concentration associated with a minimally adverse effect. Finally, quantal presentation of 

data does not allow for interpretations related to intra-rat and intra-group variability in 

performance. Due to these reporting limitations, impaired neuromuscular function and 

coordination, measured as performance on the rotarod apparatus, was excluded from 

consideration for derivation of the RfC for 1,2,3-TMB.

Endpoints carried forward for derivation of an RfC for 1,2,3-TMB, along with their 

exposure ranges and NOAEL and LOAEL values (identified by EPA), are graphically 

represented in Figure 1-1.
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Solid lines represent range of exposure concentrations. (a) Korsak and Rydzyński (1996); (b) Korsak 

et al. (2000b)

Figure 1-1. Exposure response array for endpoints resulting from 

inhalation exposure to 1,2,3-TMB considered for the derivation of the 

RfC

1.1.2. Methods of Analysis for Reference Concentration Derivation for 1,2,3-TMB 

As discussed above, endpoints observed in Korsak et al., (2000b) and Korsak and 

Rydzyński (1996) that demonstrated statistically significant (at least at the p < 0.05 level) 

pair-wise increases or decreases relative to control for at least one exposure group were 

considered for the derivation of the RfC for 1,2,3-TMB; these effects are listed in Table 1-2

above. This assessment used the BMD approach, when possible, to estimate a POD for the 

derivation of an RfC for 1,2,3-TMB (see the Supplemental Information for detailed 

methodology). The BMD approach involves fitting a suite of mathematical models to the 

observed dose-response data using EPA’s BMDS (version 2.2). Each fitted model estimates 

a BMD and its associated BMDL corresponding to a selected BMR. For continuous data (i.e., 

decreased pain sensitivity, decreased RBCs, decreased segmented neutrophils, increased 

lymphocytes, increased reticulocytes) from the Korsak and Rydzyński (1996) and Korsak 

et al. (2000b) studies, no information is available regarding the change in these responses 

that would be considered biologically significant, and thus a BMR equal to a change in the 

100

1000

10000

↓ 

pain 

sensitivity 

(a)

↓ 

RBCs

males

(b)

↑ 

lung

lesions

(b)

↓ 

Seg.

Neutrophils

males 

(b)

↓ 

Seg.

Neutrophils

females 

(b)

↑

Lymphocytes

males

(b)

↑

Lymphocytes

females

(b)

↑ 

reticulocytes

(b)

Doses

NOAEL

LOAEL
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mean equal to 1 standard deviation of the model estimated control mean was used in 

modeling the endpoints, consistent with the Benchmark Dose Technical Guidance (U.S. EPA, 

2000). The estimated BMDL is then used as the POD for deriving the RfC.

The suitability of the above methods to determine a POD is dependent on the nature of the 

toxicity database for a specific chemical. Some endpoints for 1,2,3-TMB were not amenable 

to BMD modeling for a variety of reasons, including responses only in the high exposure

group with no significant changes in responses in lower exposure groups (e.g., decreased 

RBCs) and absence of incidence data (e.g., increased inflammatory lung lesions). Increased 

lymphocytes were excluded from analysis by BMD modeling due to the unusually high 

standard deviations reported in the high exposure group. In cases where BMD modeling 

was not feasible, the NOAEL/LOAEL approach was used to identify a POD. Additionally, for 

decreased pain sensitivity, the reported SD of 3.4 in the high exposure group resulted in an 

inability of the variance power model to fit the data adequately. For this reason, the high 

dose was dropped in order to facilitate model fitting. Detailed modeling results are 

provided in the Supplemental Information.

Because an RfC is a toxicity value that assumes continuous human inhalation exposure over 

a lifetime, data derived from inhalation studies in animals need to be adjusted to account 

for the noncontinuous exposures used in these studies. In the Korsak et al., (2000b) and 

Korsak and Rydzyński (1996) studies, rats were exposed to 1,2,3-TMB for 6 hours/day, 5 

days/week for 3 months. Because no PBPK model exists for 1,2,3-TMB, the durationadjusted PODs for neurological effects in rats were calculated as follows:

PODADJ (mg/m3) = POD (mg/m3) × hours exposed per day/24 hours × days exposed 

per week/7 days

Therefore, for decreased pain sensitivity from Korsak and Rydzyński (1996), the PODADJ

would be calculated as follows:

PODADJ (mg/m3) = 97.19 mg/m3× 6 hours/24 hours × 5 days/7 days

PODADJ (mg/m3) = 17.36 mg/m3

The calculated PODADJ (mg/m3) values for all neurological, hematological, and respiratory

endpoints considered for RfC derivation are presented in Table 1-3.
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Table 1-3. Summary of dose-response analysis and point of departure 

estimation for endpoints resulting from subchronic inhalation 

exposure to 1,2,3-TMB 

Reference Endpoint Species/

Sex

POD

Basis

Best-fit 

Model; BMR

Candidate 

POD 

(mg/m3)

Candidate 

PODADJ a

(mg/m3)

Neurological Endpoints

Korsak and 

Rydzyński (1996)

Decreased pain 

sensitivity Rat, male BMDL Linear; 1 SD 97.19 17.36

Hematological Endpoints

Korsak et al. 

(2000b)

Decreased RBCs Rat, male NOAEL n/ab 523 93.39

Decreased 

segmented 

neutrophils

Rat, male BMDL Exponential 

2; 1 SD 534.81 95.50

Rat, female BMDL Hill; 1 SD 99.21 17.72

Increased 

lymphocytes

Rat, male NOAEL n/a 523 93.39

Rat, female NOAEL n/a 523 93.39

Increased

reticulocytes Rat, male BMDL Linear; 1 SD 652.90 116.58

Respiratory Endpoints

Korsak et al. 

(2000b)

Increased

inflammatory 

lung lesions

Rat, female NOAEL n/a 128 22.86

a Duration adjusted PODADJ (mg/m3) = POD × (6 hours/24 hours) × (5 days/week) in accordance with EPA 

guidance (U.S. EPA, 2002).

b No model was able to fit data adequately, or data were deemed insufficient for BMD modeling

1.1.3. Derivation of Reference Concentrations for 1,2,3-TMB 

Because the majority of selected endpoints for consideration as the critical effect 

(decreased pain sensitivity, decreased RBCs, decreased segmented neutrophils, increased 

lymphocytes, increased reticulocytes) result primarily from systemic distribution of 1,2,3-

TMB, and no available PBPK model exists for 1,2,3-TMB, the human equivalent 

concentration (HEC) for 1,2,3-TMB was calculated by the application of the dosimetric 

adjustment factor (DAF) for systemically acting gases (i.e., Category 3 gases), in accordance 

with the U.S. EPA RfC Methodology (U.S. EPA, 1994b). Additionally, although the 

observation of lung lesions would normally indicate portal-of-entry effects, various factors 

support the conclusion that 1,2,3-TMB is a systemically-acting toxicant, including the 

isomer’s relatively low water-solubility and non-reactivity as well as the observation that 

the majority of 1,2,3-TMB-induced effects are systemic in nature. Gases with these 

properties are expected to preferentially distribute to the lower regions of the respiratory 

tract where larger surface areas and thin alveolar-capillary boundaries facilitate uptake. 

Respiratory absorption of 1,2,3-TMB into the bloodstream has been observed to be 
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relatively high (~60% dose) following inhalation exposures to humans (Järnberg et al., 

1996). Therefore, increased inflammatory lung lesions are assumed to result from systemic 

distribution of 1,2,3-TMB in the bloodstream of exposed animals. DAFs are ratios of animal 

and human physiologic parameters, and are dependent on the nature of the contaminant 

(particle or gas) and the target site (e.g., respiratory tract or remote to the portal-of-entry

[i.e., systemic]) (U.S. EPA, 1994b). For gases with systemic effects, the DAF is expressed as 

the ratio between the animal and human blood:air partition coefficients:

DAF = (Hb/g)A/(Hb/b)H

DAF = 62.6/66.5

DAF = 0.94

where:

(Hb/g)A = the animal blood:air partition coefficient

(Hb/g)H = the human blood:air partition coefficient

In cases where the animal blood:air partition coefficient is lower than the human value 

(Meulenberg and Vijverberg, 2000; Järnberg and Johanson, 1995), resulting in a DAF < 1, 

the calculated value is used for dosimetric adjustments (U.S. EPA, 1994b). For example, the 

HEC for decreased pain sensitivity reported in Korsak and Rydzyński (1996) is calculated 

as follows:

PODHEC = PODADJ (mg/m3) × DAF

PODHEC = PODADJ (mg/m3) × 0.94

PODHEC = 17.36 mg/m3 × 0.94

PODHEC = 16.32 mg/m3

Table 1-4 presents the calculated HECs for the candidate critical effects, selected 

uncertainty factors (UFs) and the resulting derivation of candidate RfCs from the two

subchronic toxicity studies (Korsak et al., 2000b; Korsak and Rydzyński, 1996). 
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Table 1-4. Candidate PODADJ values, human equivalent concentrations 

(HECs), and applied uncertainty factors used in the derivation of 

Candidate RfCs for 1,2,3-TMB 

Reference Endpoint PODADJ 

(mg/L)

HEC 

(mg/m3)

a

Uncertainty Factors (UF) Candidate 

RfC 

(mg/m3) UFA UFH UFL UFS UFD UFTOTAL b

Neurological Endpoints

Korsak and 

Rydzyński 

(1996)

Decreased pain 

sensitivity 17.36 16.32 3 10 1 10 3 1,000 1.63 × 10-2

Hematological Effects

Korsak et al. 

(2000b)

Decreased RBCs, 

males 93.39 87.79 3 10 1 10 3 1,000 8.78 × 10-2

Decreased 

segmented 

neutrophils, males

95.50 89.77 3 10 1 10 3 1,000 8.98 × 10-2

Decreased 

segmented 

neutrophils, 

females

17.72 16.66 3 10 1 10 3 1,000 1.67 × 10-2

Increased 

lymphocytes, 

males

93.39 87.79 3 10 1 10 3 1,000 8.78 × 10-2

Increased 

lymphocytes, 

females

93.39 87.79 3 10 1 10 3 1,000 8.78 × 10-2

Increased 

reticulocytes, 

males

116.58 109.58 3 10 1 10 3 1,000 1.10 × 10-1

Respiratory Effects

Korsak et al. 

(2000b)

Increased 

inflammatory lung 

lesions, females

22.86 21.49 3 10 1 10 3 1,000 2.15 × 10-2

a Human equivalent concentration

b As calculated by application of uncertainty factors, not rounded.

Neurotoxicity is the most consistently observed endpoint in the toxicological database for 

1,2,3-TMB. According to EPA’s Guidelines for Neurotoxicity Risk Assessment (1998), many 

neurobehavioral changes are regarded as adverse, and the observation of correlated and 

replicated measures of neurotoxicity strengthen the evidence for a hazard. Decreased pain 

sensitivity, as measured as latency to paw-lick, is a measure of nociception (i.e., decreased 

pain sensitivity) and therefore this endpoint represents an alteration in neurobehavioral 

function (U.S. EPA, 1998). Decreased pain sensitivity was observed in two studies 

investigating short-term and subchronic exposure durations (Wiaderna et al., 1998; Korsak 

and Rydzyński, 1996), and in the presence of other metrics of altered neurobehavior, 

including impaired neuromuscular function and coordination and altered cognitive 
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function. Additionally, neurotoxicological endpoints (e.g., hand tremble, weakness) were

observed in human worker populations exposed to complex VOC mixtures containing 1,2,3-

TMB; indicating consistency and coherency of effects in humans and animals following 

exposure to 1,2,3-TMB. 

The U.S. EPA’s Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998) note that effects 

that are reversible in minutes, hours, or days after the end of exposure and appear to be 

associated with the pharmacokinetics of the agent and its presence in the body may be of 

less concern than effects that persist for longer periods of time after the end of exposure. 

Pain sensitivity was observed to return to control levels two weeks after termination of 

subchronic exposure (Korsak and Rydzyński, 1996). However, in short-term studies of 

TMBs, there is evidence indicating that decreased pain sensitivity associated with exposure 

to TMBs is not rapidly reversible and not associated with clearance of the chemical from 

the body. TMB isomers have been observed to clear rapidly from blood and nervous 

tissues (Section B.2, Appendix B), and decreased pain sensitivity persisted for up to 50-51 

days after termination of short-term exposures (Wiaderna et al., 1998). Short-term 

neurotoxicity studies of the related 1,2,4-TMB isomer also observed a persistence of 

decreased pain sensitivity after termination of exposure (Gralewicz and Wiaderna, 2001; 

Gralewicz et al., 1997). Taken as a whole, the database does not support the 

characterization of decreased pain sensitivity associated with exposure to 1,2,3-TMB as 

rapidly reversible upon clearance from the body. Given the consistency of decreased pain 

sensitivity across independent studies and multiple durations of exposure in animal 

studies, and the consistency of observed neurotoxicity in animals and humans, EPA 

concluded that there is strong evidence that neurotoxicity is a toxicity hazard associated 

with exposure to 1,2,3-TMB. Further, EPA concluded that decreased pain sensitivity is an 

adverse effect and is the most appropriate effect on which to base the RfC. Therefore, the 

candidate RfC for neurotoxicity based on decreased pain sensitivity was selected as 

the RfC for 1,2,3-TMB. 

A PODHEC of 16.3 mg/m3 for decreased pain sensitivity (Korsak and Rydzyński, 1996) was 

used as the POD to derive the chronic RfC for 1,2,3-TMB. The uncertainty factors (UFs), 

selected and applied in accordance with the procedures described in EPA’s A Review of the 

Reference Dose and Reference Concentration Processes (2002) (Section 4.4.5 of the report), 

address five areas of uncertainty resulting in a composite UF of 1,000. This composite UF

was applied to the selected POD to derive an RfC. 

An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3) was applied to 

account for uncertainty in characterizing the toxicokinetic and toxicodynamic differences 
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between rats and humans following inhalation exposure to 1,2,3-TMB. In this assessment, 

the use of a DAF to extrapolate external exposure concentrations from rats to humans 

reduces toxicokinetic uncertainty in extrapolating from the rat data, but does not account 

for the possibility that humans may be more sensitive to 1,2,3-TMB than rats due to 

toxicodynamic differences. A default UFA of 3 was thus applied to account for this 

remaining toxicodynamic and residual toxicokinetic uncertainty.

An intraspecies uncertainty factor, UFH, of 10 was applied to account for potentially 

susceptible individuals in the absence of data evaluating variability of response in the 

human population following inhalation of 1,2,3-TMB. No information is currently available 

to predict potential variability in human susceptibility, including variability in the 

expression of enzymes involved in 1,2,3-TMB metabolism. 

A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because the current approach 

is to address this factor as one of the considerations in selecting a BMR for BMD modeling. 

In this case, a BMR equal to a change in the mean equal to 1 standard deviation of the 

model estimated control mean for decreased pain sensitivity was selected under the 

assumption that this BMR represents a minimal, biologically significant change for this 

endpoint. 

A subchronic to chronic uncertainty factor, UFS, of 10 was applied to account for 

extrapolation from a subchronic exposure duration study to derive a chronic RfC. The 10-

fold uncertainty factor is applied to the POD identified from the subchronic study on the 

assumption that effects observed in a similar chronic study would be observed at lower 

exposure concentrations for a number of possible reasons, including potential cumulative 

damage occurring over the duration of the chronic study or an increase in the magnitude or 

severity of effect with increasing duration of exposure. 

A database uncertainty factor, UFD, of 3 (101/2 = 3.16, rounded to 3) was applied to account 

for database deficiencies. Strengths of the database include the two well-designed 

subchronic studies that observe exposure-related effects in multiple organ systems 

(nervous, hematological, and respiratory systems) in Wistar rats exposed to 1,2,3-TMB via 

inhalation. However, the database lacks a multi-generational reproductive toxicity study 

and a developmental toxicity study investigating effects due to 1,2,3-TMB exposure. 

Normally, the lack of both of these types of studies in a toxicity database would warrant the 

application of a 10-fold UFD in accordance with EPA’s Review of the Reference Dose and 

Reference Concentration Processes (2002). Although there is no developmental toxicity 

study for 1,2,3-TMB, Saillenfait et al. (2005) investigated the developmental toxicity of the 
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other two TMB isomers (1,2,4-TMB and 1,3,5-TMB) and observed developmental toxicity at 

levels much higher than those eliciting neurotoxicity, hematotoxicity, and respiratory

toxicity in adult animals (Korsak studies). Given that toxic effects were observed at lower 

concentrations in adult rats exposed to 1,2,4-TMB and 1,3,5-TMB compared with rats

exposed in utero and the similarities in toxicity profiles amongst the three isomers, it is 

unlikely that the inclusion of a developmental toxicity study for 1,2,3-TMB would result in a 

POD that is lower than the POD associated with neurotoxicity for this isomer. Thus, the 

application of an UF to account for the lack of a development toxicity study is not 

warranted.

Although, a multi-generation reproductive study does not exist for 1,2,3-TMB, there is a 

multi-generation developmental/reproductive study for high flash naphtha, of which 1,2,3-

TMB is a constituent. This study demonstrates effects on postnatal growth at lower 

exposures in the F3 generation (2,460 mg/m3) compared to the F2 or F1 generation (7,380 

mg/m3) (McKee et al., 1990), but did not observe a consistent effect on reproductive 

parameters. This raises some concern that addition of a multi-generational reproductive 

study for 1,2,3-TMB might result in the identification of a lower POD.

EPA’s Review of the Reference Dose and Reference Concentration Processes (2002) also 

recommends that the database uncertainty factor take into consideration whether there is 

concern from the available toxicity database that the developing organism may be 

particularly susceptible to effects in specific organ systems. TMBs (unspecified isomer) are 

able to cross the placenta (Cooper et al., 2001; Dowty et al., 1976); therefore, as 

neurotoxicity is observed in adult animals, there is concern that exposure to 1,2,3-TMB 

may result in neurotoxicity in the developing organism. EPA’s Guidelines for Neurotoxicity 

Risk Assessment (1998) identify specific effects observed in adult animals (e.g., cognitive 

and motor function) as effects that can also affect the developing organism exposed in 

utero. EPA’s Guidelines for Neurotoxicity Risk Assessment (1998) also indicate that 

neurotoxicants may have greater access to the nervous system in developing organisms 

due to an incomplete blood-brain barrier and immature metabolic detoxifying pathways. 

Therefore, there is some concern that the lack of a developmental neurotoxicity study is a 

deficiency in the database and that the inclusion of such a study would potentially result in 

a lower POD than the POD for neurotoxicity identified from the available 1,2,3-TMB toxicity 

database. In summary, a 3-fold database UF was applied to account for the lack of both a 

multi-generation reproductive study and a developmental neurotoxicity study in the 

available database for 1,2,3-TMB. 
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Application of this 1000-fold composite UF to the PODHEC yields the following chronic RfC 

for 1,2,3-TMB:

RfC = PODHEC ÷ UF 

RfC = 16.3 mg/m3 ÷ 1,000 

RfC = 0.02 mg/m3 = 2 × 10-2 mg/m3 (rounded to one significant digit)

1.1.4. Uncertainties in the Derivation of the Reference Concentration for 1,2,3-TMB 

As presented above, the UF approach following EPA practices and RfC guidance (U.S. EPA, 

2002, 1994b), was applied to the PODHEC in order to derive the chronic RfC for 1,2,3-TMB. 

Factors accounting for uncertainties associated with a number of steps in the analyses 

were adopted to account for extrapolation from animals to humans, a diverse human 

population of varying susceptibilities, POD determination methodologies (NOAEL, LOAEL, 

or BMDL), and database deficiencies.

The critical effect selected, decreased pain sensitivity, does not introduce substantial 

variability into the RfC calculation as selection of alternative hematological or respiratory 

effects would result in similar RfCs that would be equivalent when rounding to one 

significant digit (i.e., 2 × 10-2 mg/m3 , see Figure 1-2). Some uncertainty exists regarding 

the selection of the BMRs for use in BMD modeling due to the absence of information to 

determine the biologically significant level of response associated with the endpoints. 

However in cases such as this, the selection of a BMR of 1 standard deviation for 

continuous endpoints is supported by EPA guidance (U.S. EPA, 2000). Uncertainty 

regarding the selection of particular models for individual endpoints exists as selection of 

alternative models could decrease or increase the estimated POD and consequently, the 

RfC. The selection criteria for model selection was based on a practical approach as 

described in EPA’s BMD Technical Guidance (U.S. EPA, 2000). Uncertainty may exist in the 

default dosimetry methods used to calculate HEC estimates, but such uncertainties would 

apply equally to all endpoints. 

1.1.5. Confidence Statement for the Reference Concentration for 1,2,3-TMB 

Confidence levels of high, medium, or low are assigned to the study used to derive the RfC, 

the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA’s Methods for 

Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry

(U.S. EPA, 1994b). Confidence in the principal study from which the critical effect was 

identified, Korsak and Rydzyński (1996) is medium. The study is a well-conducted 

peer-reviewed study that utilized three dose groups plus untreated controls, and an 
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appropriate number of animals per dose group, and performed statistical analyses. One 

area of uncertainty regarding this study is the lack of reported actual exposure 

concentrations. However, as the methods by which the test atmosphere was generated and 

analyzed were reported in sufficient detail, and given the fact that this laboratory has used 

this methodology in subsequent studies (Korsak et al., 2000b) and achieved appropriate 

actual concentrations (i.e., within 10% of target concentrations), the concern regarding the 

lack of reported actual concentrations is minimal. The critical effect on which the RfC is 

based is well-supported as the evidence for 1,2,3-TMB-induced neurotoxicity is consistent

and coherent across multiple animals species (i.e., human and rat) and across multiple 

exposure durations (i.e., acute, short-term, and subchronic) (Lutz et al., 2010; Wiaderna et 

al., 1998; Korsak and Rydzyński, 1996). Confidence in the database for 1,2,3-TMB is low 

to medium as the database includes acute, short-term, and subchronic toxicity studies in 

rats, but lacks chronic, multi-generation reproductive, developmental toxicity, and

developmental neurotoxicity studies. Additionally, the studies supporting the critical effect 

predominantly come from the same research institute. Overall confidence in the RfC for 

1,2,3-TMB is low to medium. 

1.1.6. Comparison of Candidate Reference Concentrations for 1,2,3-TMB 

The predominant noncancer effect observed following acute, short-term, and subchronic 

inhalation exposures to 1,2,3-TMB is neurotoxicity, although respiratory toxicity is also 

observed following acute and subchronic exposures, while hematological effects are 

observed after subchronic exposures. Figure 1-2 provides a graphical display of all 

candidate PODs and RfCs derived from the two subchronic studies considered in the 

selection of the inhalation RfC for 1,2,3-TMB. 
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Figure 1-2. Array of candidate PODHEC values with applied UFs and 

candidate RfCs for neurological, respiratory, and hematological effects 

resulting from inhalation exposure to 1,2,3-TMB 

1.2. Oral Reference Dose for Effects other than Cancer for 1,2,3-TMB 

The available oral database is inadequate to derive an RfD for 1,2,3-TMB. No chronic, 

subchronic, or short-term oral exposure studies were found in the literature. However, as 

discussed in Section 1, there are sufficient similarities between isomers regarding observed 

toxicological effects that support adopting the RfD for 1,2,4-TMB as the RfD for 1,2,3-TMB. 

Specifically, the qualitative pattern of neurotoxic effects following short-term and 

subchronic inhalation exposures is similar between isomers. Particularly important to this 

determination is that, although 1,2,3-TMB is observed to decrease pain sensitivity at lower 

concentrations than 1,2,4-TMB (LOAEL values of 123 vs. 492 mg/m3), the magnitude of 

decreased pain sensitivity is similar for 1,2,4-TMB and 1,2,3-TMB, especially in the low and 

mid exposure concentrations. This similarity of effect in the low dose region of the doseresponse curve is exhibited by equal RfC values derived from isomer-specific data: 2 × 10-2

mg/m3. Although a PBPK model exists for 1,2,4-TMB that allows for route-to-route 

extrapolation from inhalation to oral exposure, no such model exists for 1,2,3-TMB. 

0.01

0.1

1

10

100

Decreased 

pain 

sensitivity

Increased 

inflammatory 

lung lesions

Decreased 

RBCs

Decreased

Segmented

Neutrophils

males

Decreased

Segmented

Neutrophils

females

Increased

Lymphocytes

males

Increased

Lymphocytes

females

Decreased 

Reticulocytes

Neurological

Effects

Respiratory

Effects

Hematological

Effects

Database Uncertainty Subchronic to Chronic Intraspecies Variability

Interspecies Extrapolation RfC POD
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However, similarities in blood:air and tissue:air partition coefficients and degree of 

absorption into the bloodstream between the 1,2,4-TMB and 1,2,3-TMB isomers support 

the conclusion that internal blood dose metrics for 1,2,3-TMB would be similar to those 

calculated for 1,2,4-TMB using that isomer’s available PBPK model. Also, the qualitative 

metabolic profiles for the two isomers are similar, with dimethylbenzyl hippuric acids 

being the major terminal metabolite for both isomers, such that first-pass metabolism 

through the liver is not expected to differ greatly between 1,2,4-TMB and 1,2,3-TMB. 

Therefore, given the above similarities in toxicokinetics and toxicity, the RfD derived 

for 1,2,4-TMB, 6 × 10-3 mg/kg-day, was adopted for the RfD for 1,2,3-TMB.

1.2.1. Uncertainties in the Derivation of the Reference Dose for 1,2,3-TMB

The uncertainties regarding adopting the RfD for 1,2,4-TMB as the RfD for 1,2,3-TMB 

encompass previous areas of uncertainty involved in the derivation of the RfD for 1,2,4-

TMB (see Section 2). Additionally, there is uncertainty in this adoption regarding the 

assumptions made about the similarity in toxicity and toxicokinetics between the two 

isomers. However, as discussed above and in Sections 1 and in B.2, there is strong evidence 

that both isomers share multiple commonalities and similarities regarding their 

toxicokinetic and toxicological properties that support adopting one isomer’s value for the 

other. 

1.2.2. Confidence Statement for the Reference Dose for 1,2,3-TMB

As noted previously, a confidence level of high, medium, or low is assigned to the study 

used to derive the RfD, the overall database, and the RfD itself, as described in U.S. EPA 

(1994b), Section 4.3.9.2. The chronic RfD of 6 × 10-3 mg/kg-day derived for 1,2,4-TMB was 

adopted as the RfD for 1,2,3-TMB based on the conclusion that the two isomers were 

sufficiently similar regarding chemical properties, kinetics, and toxicity. Thus, confidence 

in the study from which the critical effect was identified, Korsak and Rydzyński 

(1996) is medium (see above). Confidence in the database is low to medium as the 

database includes acute, short-term, and subchronic studies in rats and mice. The database 

lacks a multi-generation reproductive, developmental toxicity, or developmental 

neurotoxicity study, and the studies supporting the critical effect predominantly come from 

the same research institute. Overall confidence in the RfD for 1,2,3-TMB is low due to 

uncertainties surrounding the adoption of the RfD derived for 1,2,4-TMB as the RfD for 

1,2,3-TMB.
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1.3. Cancer Assessment for 1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB

Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (2005), the database for 

1,2,4-TMB, 1,2,3-TMB, and 1,3,5-TMB provides “inadequate information to assess the 

carcinogenic potential”. This characterization is based on the limited and equivocal 

genotoxicity findings, and the lack of data indicating carcinogenicity in experimental animal 

species. Information available on which to base a cancer assessment is lacking, and thus, 

no cancer risk values are derived.
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